Pyrazolo Pyrimidine Derivatives and Methods of Use Thereof

ABSTRACT

This invention generally relates to pyrazolo pyrimidine derivatives useful as, inter alia, inhibitors of short chain dehydrogenase/reductase (SDR) family of NAD(P)(H) dependent oxido-reductases. More specifically, the invention relates to pyrazolo pyrimidine derivatives, including derivatives and analogs of SDR inhibitors, pharmaceutical compositions containing derivatives and analogs of SDR inhibitors, methods of making derivatives and analogs of SDR inhibitors and methods of use thereof.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.12/194,469, filed Aug. 19, 2008, which is a continuation of U.S. patentapplication Ser. No. 10/871,732, filed Jun. 18, 2004, (now U.S. Pat. No.7,429,596) which claims priority to U.S. Application No. 60/480,501,filed Jun. 20, 2003, the entire disclosure of which is incorporatedherein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support by Grant Nos. AI44009and NCRR RR01614 awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

FIELD

This invention generally relates to pyrazolo pyrimidine derivatives,including derivatives and analogs of inhibitors of short chaindehydrogenase/reductase (SDR) family of NAD(P)(H) dependentoxido-reductases. More specifically, the invention relates to pyrazoloand pyrollo pyrimidine derivatives, including derivatives and analogs ofSDR inhibitors, pharmaceutical compositions containing the pyrazolopyrimidine derivatives, and methods of making and methods of usethereof.

BACKGROUND

Cancer of the lung and bronchus (lung cancer) is the second most commoncancer among both men and women and is the leading cause of cancer deathin both sexes. Among men, age-adjusted lung cancer incidence rates (per100,000) range from a low of about 14 to a high of 117, an eight-folddifference, depending upon ethnicity. The rates among men are about twoto three times greater than the rates among women in each of theracial/ethnic groups.

Leukemia and lymphoma are the most common fatal cancers in young menunder age 39. Leukemia, Hodgkin and non-Hodgkin lymphoma and myeloma arecancers that originate in the bone marrow or lymphatic tissues. Anestimated 106,300 people in the United States will be diagnosed withleukemia, lymphoma or myeloma in 2002. New cases of leukemia, Hodgkinand non-Hodgkin lymphoma and myeloma account for 8.3 percent of the1,284,900 new cancer cases diagnosed in the United States this year. SeeSurveillance, Epidemiology and End Results (SEER) Program 1979-1998,National Cancer Institute; American Cancer Society.

An estimated 616,695 Americans are currently living with leukemia,Hodgkin and non-Hodgkin lymphoma and myeloma. Leukemia, lymphoma andmyeloma will cause the deaths of an estimated 58,300 people in theUnited States this year. These blood cancers will account for nearly10.5 percent of the deaths from cancer in 2002 based on the total of555,500 cancer-related deaths (all sites).

The short chain dehydrogenase/reductase (SDR) family of NAD(P)(H)dependent oxido-reductases are believed to have a role in disease, forexample, cancer, inflammatory disease, and diabetes. The SDR familyrepresents a diverse family of >63 human proteins (Oppermann, U. C., etal., Chem Biol Interact, 130-132: 699-705, 2001. Kallberg, Y., et al.,Eur J Biochem, 269: 4409-17, 2002. Kallberg, Y., et al., Protein Sci,11: 636-41, 2002). These enzymes are responsible for the oxidation orreduction of a wide range of endogenous (prostaglandins, steroidhormones, retinal, dihydropteridin, UDP, and trans 2-enoyl CoA) andexogenous chemicals (anthracyclin drugs, quininones, and others). TheSDR family members thus control the cell specific production/destructionof potent hormones as well as the detoxification of important classes ofdrugs such as the anti-cancer agent adriamycin (Forrest, G. L. et al.,Chem Biol Interact, 129: 21-40, 2000).

Carbonyl reductase (CBR) (NADPH: secondary-alcohol oxidoreductase) ispart of a group of NADPH-dependent cytosolic enzymes called short chaindehydrogenase/reductase (SDR) that catalyze the reduction of variouscarbonyl compounds to their corresponding alcohols. The enzyme isubiquitous in nature and acts on a large number of biologically andpharmacologically active compounds. Carbonyl reductase is believed tofunction physiologically as a dehydrogenase or reductase ofprostaglandins or hydroxysteroids, as well as in drug metabolism.

Carbonyl reductase is primarily monomeric in structure, and has beencharacterized in humans from placenta, liver, and breast tissue. CBRbears a low overall degree of homology (24-36%) with other SDR enzymesfrom mammalian sources such as mouse and pig (Nakanishi, M. et al.Biochem. Biophys. Acta 194: 1311-16, 193). However, all of these enzymesare linked by two common consensus sequences; the sequence TGxxxGxG,found in the N-terminal portion of the molecule and responsible forbinding the NADPH co-enzyme, and the sequence YxxxK, located close tothe C-terminal end of the molecule, and active in carbonyl reduction.Differences in amino acid sequences between these enzymes can beresponsible, in part, for differences in their respective substratespecificities for various carbonyl compounds.

The bioreduction of prostaglandin (PGE) by carbonyl reductase serves toregulate cellular levels of PGE. A wide variety of biological activitiesare ascribed to PGEs including smooth muscle contraction, plateletaggregation, inflammation, inhibition of insulin secretion, andlymphocyte function. Excessive PGE production is associated withinflammatory diseases, diabetes, and suppression of the immune responseInhibitors of PGE biosynthesis, such as indomethacin and ibuprofen, arecommonly used to treat inflammation and inflammatory diseases anddepressed cellular immunity in patients with conditions such asHodgkin's disease (Isselbacher K. J. et al. Harrison's Principles ofInternal Medicine, Vol. 1: 431-435, 1994, McGraw-Hill, New York City).

In human liver, carbonyl reductase also reduces quinones, an importantclass of mutagens and carcinogens, and appears to be the principlemechanism for detoxification of these compounds. CBR production isstimulated by carcinogens such as butyl hydroxyanisole andbeta-naphthoflavone that also induce other cancer-protective enzymes(Forrest, G. L. et al. Biochim. Biophys. Acta 1048: 149-55, 1990).

Human carbonyl reductase 1 (CBR1) has been characterized as havingsimilarity to carbonyl reductases from porcine lung (GI 416425), mouseadipocytes (GI 50004), and human liver (GI 118519). Human CBR1 is 85%identical to porcine carbonyl reductase. The role of carbonyl reductasein cells is not understood.

Carbonyl reductase is also involved in the metabolism of anthracyclines,a widely used class of anticancer chemotherapeutic drugs. Daunorubicin(DNR) and Doxorubicin (DXR), the two principle anthracyclines used incancer chemotherapy, are reduced to their respective alcohols bycarbonyl reductase. The alcohol products are much less effectiveantitumor agents than the parent compounds. In fact, increased carbonylreductase levels associated with some anthracycline resistant tumorssuggest that increased carbonyl reductase activity may be responsiblefor drug resistance in these cells (Soldan, M. et al. Biochem.Pharmacol. 51: 116-23, 1996; Gonzalez, B. et al. Cancer Res. 55:4646-50, 1995). Another problem of anthracyclines is cardiotoxicity, butthe causative agents are suggested to be the alcohol products ofcarbonyl reductase catalyzed reaction. (Forrest, G. L. et al., Chem BiolInteract, 129: 21-40, 2000.)

Daunorubicin is one of the family of anthracycline antibiotic drugs thatinclude daunorubicin, doxorubicin, epirubicin, and idarubicin. Thesedrugs are used in the treatment of acute leukemia, lymphomas, andmyeloma. Daunorubicin is used to treat acute myeloid leukemia, acutelymphocytic leukemia, chronic myelogenous leukemia, neuroblastoma.Liposomal daunorubicin belongs to the general group of medicines knownas antineoplastics. It is used to treat advanced acquiredimmunodeficiency syndrome (AIDS)-associated Kaposi's sarcoma (KS),

Molecules that inhibit short chain dehydrogenase/reductase (SDR) familyof NAD(P)(H) dependent oxido-reductases, for example, carbonylreductase, satisfy a need in the art by providing new diagnostic ortherapeutic compositions useful in the prevention and treatment ofinflammation, immunological disorders, cancer, and drug resistance incancerous cells, and reducing toxicity associated with CBR catalyzedmetabolites of known drugs.

SUMMARY

The invention is generally related to inhibitors of short chaindehydrogenase/reductase (SDR) family of NAD(P)(H) dependentoxido-reductases, and derivatives and analogs thereof. The inventionfurther relates to pharmaceutical compositions containing the inhibitorsof SDR family of NAD(P)(H) dependent oxido-reductases, and derivativesand analogs thereof, methods of making the inhibitors of SDR family ofNAD(P)(H) dependent oxido-reductases and derivatives and analogsthereof, and methods of use thereof.

In one embodiment, the present invention is directed to a compound ofFormula I or II:

or a pharmaceutically-acceptable salt or prodrug thereof;

wherein:

-   -   Y is N or CR₅;    -   Z is NR₃R₄, halo, H, OH, alkyl, alkyloxy, or haloalkyl;    -   R_(1a) is indolyl, thiazolyl, benzyl, biphenylyl, thiophenyl,        pyrrolyl, or phenyl, wherein said phenyl is substituted with at        least one of OH, —NR₃R₄, —C(═O)NR₆R₇, —CN, NO₂—C(═O)OH,        —C(═O)O-alkyl, (C₁-C₄)alkyl, halo, haloalkyl or haloaryl; and        wherein said indolyl, thiazolyl, benzyl, biphenylyl, thiophenyl,        or pyrrolyl is optionally substituted with OH, —NR₃R₄,        —C(═O)NR₆R₇, —CN, NO₂, —C(═O)O—R₃, (C₁-C₄)alkyl, halo, haloalkyl        or haloaryl;    -   R_(1b) is indolyl, thiazolyl, benzyl, biphenylyl, thiophenyl,        pyrrolyl, or phenyl wherein said indoyl, thiazolyl, benzyl,        biphenylyl, thiophenyl, pyrrolyl, phenyl is optionally        substituted with —OH, —NR₃R₄, —C(═O)NR₆R₇, —CN, NO₂, —C(═O)O—R₃,        (C₁-C₄)alkyl, halo, haloalkyl, or haloaryl;    -   R₂ is C₁-C₆ alkyl or C₄-C₇ cycloalkyl, wherein said alkyl or        said cycloalkyl is optionally substituted with mono- or        di-alkoxy, mono- or di-halophenyl, mono- or di-(C₁₋₄)alkoxy        phenyl, mono- or di-(C₁₋₄)alkyl phenyl, perhalo(C₁₋₄)alkyl        phenyl, carboxyl, tert-butyl carboxyl, phosphoryl, (C₁₋₆)alkyl,        (C₄₋₇)cycloalkyl, indolyl, isoindolyl, pyridyl, naphthyl,        pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl,        pyrazinyl, pyridazinyl, furyl, thienyl, or alkylmorpholino;    -   R₃ and R₄ are independently H, C₁-C₆ alkyl, t-Boc,        morpholino(C₁-C₄)alkyl, carboxy(C₁-C₃)alkyl,        (C₁-C₄)alkoxycarbonyl(C₁-C₃)alkyl, aryl, heteroaryl, aryloxy,        heterocycle, cycloalkyl, alkenyl with the proviso that the        double bond of the alkenyl is not present at the carbon atom        that is directly linked to N, alkynyl with the proviso that the        triple bond of the alkynyl is not present at the carbon atom        that is directly linked to N, perfluoroalkyl, —S(O)₂alkyl,        —S(O)₂aryl, —(C═O)heteroaryl, —(C═O)aryl, —(C═O)(C₁-C₆) alkyl,        —(C═O)cycloalkyl, —(C═O)heterocycle, alkyl-heterocycle, aralkyl,        arylalkenyl, —CONR₆R₇, —SO₂R₆R₇, arylalkoxyalkyl,        arylalkylalkoxy, heteroarylalkylalkoxy, aryloxyalkyl,        heteroaryloxyalkyl, aryloxyaryl, aryloxyheteroaryl,        alkylaryloxyaryl, alkylaryloxyheteroaryl, alkylaryloxyalkyamine,        alkoxycarbonyl, aryloxycarbonyl, or heteroaryloxycarbonyl;    -   R₅ are independently H, —OH, halo, optionally monosubstituted        (C₁-C₆)alkyl, optionally monosubstituted (C₁-C₄)alkoxycarbonyl,        optionally monosubstituted (C₁-C₄)alkanoyl, carbamoyl,        optionally monosubstituted (C₁-C₄)alkyl carbamoyl, phenyl,        halophenyl, optionally monosubstituted (C₁-C₄)alkylphenyl,        optionally monosubstituted (C₁-C₄)alkoxyphenyl, or optionally        monosubstituted perhalo(C₁-C₄)alkylphenyl, wherein said optional        substitution is (C₁-C₄)alkyl, OH, or halogen;    -   R₆ and R₇ are independently H, alkyl, aryl, heteroaryl,        alkylaryl, arylalkyl, heteroarylalkyl, or alkylheteroaryl;    -   provided the compound is not        1-tert-butyl-3-p-tolyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine.

In another embodiment, the present invention is directed to a method forpreventing or treating cancer in a mammal, comprising the step ofadministering to the mammal an effective amount of a compound of formulaI or formula II, or a pharmaceutically acceptable salt thereof.

In yet another embodiment, the present invention is directed to a methodfor preventing or treating a disease or condition associated withcarbonyl reductase 1 in a mammal in need thereof, comprising the stepsof administering to the mammal a composition comprising an effectiveamount of a compound of formula I or formula II, or a pharmaceuticallyacceptable salt thereof.

In yet another embodiment, the present invention is directed to a methodof preventing or treating a disease or condition associated with thesynthesis of prostaglandin E in a mammal in need thereof comprising thesteps of administering to the mammal a composition comprising aneffective amount of a compound of formula I or formula II, or apharmaceutically acceptable salt thereof, and inhibiting synthesis ofprostaglandin E2.

In a further embodiment, the present invention is directed to a methodfor preventing or treating a disease or condition associated with shortchain dehydrogenase/reductase (SDR) family of NAD(P)(H) dependentoxido-reductases in a mammal in need thereof, comprising the step ofadministering to the mammal an effective amount of a compositioncomprising an effective amount of a compound of formula I or formula II,or a pharmaceutically acceptable salt thereof.

In a further embodiment, the present invention is directed to a methodfor identifying a therapeutic cancer treatment, comprising the steps ofcontacting a tumor cell culture with an effective amount of acomposition comprising an effective amount of a compound of formula I orformula II, or a pharmaceutically acceptable salt thereof, measuringgrowth inhibition of the tumor cells in culture; and identifying atherapeutic cancer treatment for a mammalian subject by inhibition ofthe tumor cell growth in culture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the role of CBR in the biosynthesis of prostaglandins.

FIG. 2 shows enzymatic activity of 11β-hydroxysteroid dehydrogenase Iand 11β-hydroxysteroid dehydrogenase II.

FIG. 3 shows enzymatic activity of carbonyl reductase 1 (CBR1) ondaunorubicin.

FIG. 4 shows carbonyl reductase 1 (CBR1) is up-regulated in some classesof lung tumors.

FIG. 5 shows AB129 inhibits menadione reducing activity of carbonylreductase 1 (CBR1).

FIG. 6 shows kinetic analysis of carbonyl reductase 1 (CBR1).

FIG. 7 shows selection of RNAi hairpins for targeting of carbonylreductase 1 (CBR1).

FIG. 8 shows carbonyl reductase 1 (CBR1) is required for A549 lungcarcinoma cell viability.

FIG. 9 shows the structure of glycyrrizic acid.

FIG. 10 shows the structure of (1) AB129, (2) PP1, (3) AB60, and (4)AB61.

FIG. 11 shows that AB129 causes a morphological change in human lungcarcinoma cells.

FIG. 12 shows effects of AB129 on cell cycle of A549 lung carcinomacells.

FIG. 13 shows probes for an affinity experiment to identify AB129target.

FIG. 14 shows an affinity experiment using A549 lung carcinoma celllysate.

FIG. 15 shows protein identification by collision induced dissociationmass spectrometry (LC/MS/MS).

FIG. 16 shows amino acid sequence identification of trypsin digest bymass spectrometry.

FIG. 17 shows mass spectrometric identification of protein fragments.

FIG. 18 shows mass spectrometric identification of protein fragments.

FIG. 19 shows mass spectrometric identification of protein fragments.

FIG. 20 shows mass spectrometric identification of protein fragments.

FIG. 21 shows mass spectrometric identification of protein fragments.

FIG. 22 shows mass spectrometric identification of protein fragments.

FIG. 23 kinetic parameters of AB129 inhibition of carbonyl reductase 1(CBR1).

FIG. 24 shows a molecular model for docking of AB129 within porcinecarbonyl reductase 1 (CBR1).

FIG. 25 shows conserved amino acid residues in short chaindehydrogenase/reductase (SDR) enzymes.

FIG. 26 shows sequence alignment of NADPH binding pocket in SDR enzymes.

FIG. 27 shows a Rossman fold of SDR enzymes.

FIG. 28 shows effects on inhibition by mutation at Asn90 of carbonylreductase 1 (CBR1).

FIG. 29 shows Asn90 has a role for binding of AB129 to carbonylreductase 1 (CBR1).

FIG. 30 shows N90V mutant of carbonyl reductase 1 (CBR1) is lesssensitive to AB129 than wild type CBR1.

FIG. 31 shows glutathione-modified eicosanoids.

FIG. 32 shows glutathione binding activity of wild type and mutantcarbonyl reductase 1 (CBR1).

FIG. 33 shows carbonyl reductase (CBR) can cause anthracyclineresistance.

FIG. 34 shows that AB129 reinforces the cytotoxicity of daunorubicin.

FIG. 35 shows AB129 analogs display differing selectivities for carbonylreductase (CBR) and kinases.

FIG. 36 shows potential library substituents for inhibitors of SDRenzymes.

FIG. 37 shows pyrrolopyrimidine scaffold validation.

FIG. 38 shows solid phase pyrrolopyrimidine library synthesis.

FIG. 39 shows scaffold loading optimization.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

With respect to pyrazolo pyrimidine or, “derivative” refers to acompound of general formula I or II:

where the variables are as defined herein.

With respect to pyrazolo pyrimidine or AB-129 compound, “analog” or“functional analog” refers to a modified form of the respective pyrazolopyrimidine or AB-129 compound derivative in which one or more chemicallyderivatized functional substituent (R_(1a), R_(1b), R₂, R₃, R₄ or Z) ora ring atom (Y) has been modified such that the analog retainssubstantially the same biological activity or improved biologicalactivity as the unmodified pyrazolo pyrimidine or AB-129 compoundderivative in vivo and/or in vitro.

The present invention is directed to pyrazolo pyrimidine derivatives,compositions containing these derivatives, and methods of their use forthe prevention and treatment of, inter alia, cancer, metastatic cancer,inflammation, and diabetes.

The following definitions are provided for the full understanding ofterms and abbreviations used in this specification.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include the plural reference unless the context clearlyindicates otherwise. Thus, for example, a reference to “an antagonist”or “an agonist” includes a plurality of such antagonists or a pluralityof such agonists, and a reference to “a compound” is a reference to oneor more compounds and equivalents thereof known to those skilled in theart, and so forth.

The abbreviations in the specification correspond to units of measure,techniques, properties, or compounds as follows: “min” means minutes,“h” means hour(s), “μL” means microliter(s), “mL” means milliliter(s),“mM” means millimolar, “M” means molar, “mmole” means millimole(s), “cm”means centimeters, “SEM” means standard error of the mean and “IU” meansInternational Units, “° C.” means degrees Celcius. “ΔED₅₀ value” meansdose which results in 50% alleviation of the observed condition oreffect (50% mean maximum endpoint), “ΔID₅₀” means dose which results in50% inhibition of an observed condition or effect or biochemical process(50% mean maximum endpoint).

“Alkyl” refers to an optionally substituted, saturated straight,branched, or cyclic hydrocarbon having from about 1 to about 20 carbonatoms (and all combinations and subcombinations of ranges and specificnumbers of carbon atoms therein), with from about 1 to about 8 carbonatoms, herein referred to as “lower alkyl”, being preferred. Alkylgroups include, but are not limited to, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, cyclopentyl, isopentyl,neopentyl, n-hexyl, isohexyl, cyclohexyl, cyclooctyl, adamantyl,3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl. Lower alkylrefers to alkyl having 1 to 4 carbon atoms.

“Cycloalkyl” refers to an optionally substituted, alkyl group having oneor more rings in their structures having from about 3 to about 20 carbonatoms (and all combinations and subcombinations of ranges and specificnumbers of carbon atoms therein), with from about 3 to about 10 carbonatoms being preferred. Multi-ring structures can be bridged or fusedring structures. Groups include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and adamantyl.Specifically included within the definition of “cyclic alkyl” are thosealiphatic hydrocarbon chains that are optionally substituted.

“Perfluorinated alkyl” refers to an alkyl, as defined above, in whichthe hydrogens directly attached to the carbon atoms are completelyreplaced by fluorine.

“Alkenyl” refers to an alkyl group of at least two carbon atoms havingone or more double bonds, wherein alkyl is as defined herein. Alkenylgroups can be optionally substituted.

“Alkynyl” refers to an alkyl group of at least two carbon atoms havingone or more triple bonds, wherein alkyl is as defined herein. Alkynylgroups can be optionally substituted.

“Aryl” as used herein, refers to an optionally substituted, mono-, di-,tri-, or other multicyclic aromatic ring system having from about 5 toabout 50 carbon atoms (and all combinations and subcombinations ofranges and specific numbers of carbon atoms therein), with from about 6to about 10 carbons being preferred. Non-limiting examples include, forexample, phenyl, naphthyl, anthracenyl, and phenanthrenyl.

“Heteroaryl” refers to an optionally substituted, mono-, di-, tri-, orother multicyclic aromatic ring system that includes at least one, andpreferably from 1 to about 4 sulfur, oxygen, or nitrogen heteroatom ringmembers. Heteroaryl groups can have, for example, from about 3 to about50 carbon atoms (and all combinations and subcombinations of ranges andspecific numbers of carbon atoms therein), with from about 4 to about 10carbons being preferred. Non-limiting examples of heteroaryl groupsinclude, for example, pyrryl, furyl, pyridyl, 1,2,4-thiadiazolyl,pyrimidyl, thienyl, isothiazolyl, imidazolyl, tetrazolyl, pyrazinyl,pyrimidyl, quinolyl, isoquinolyl, thiophenyl, benzothienyl,isobenzofuryl, pyrazolyl, indolyl, purinyl, carbazolyl, benzimidazolyl,and isoxazolyl.

“Heterocyclic ring” refers to a stable 5- to 7-membered monocyclic orbicyclic or 7- to 10-membered bicyclic heterocyclic ring that issaturated, partially unsaturated or unsaturated (aromatic), and whichcontains carbon atoms and from 1 to 4 heteroatoms independently selectedfrom the group consisting of N, O and S and including any bicyclic groupin which any of the above defined heterocyclic rings is fused to abenzene ring. The nitrogen and sulfur heteroatoms may optionally beoxidized. The heterocyclic ring may be attached to its pendant group atany heteroatom or carbon atom that results in a stable structure. Theheterocyclic rings described herein may be substituted on carbon or on anitrogen atom if the resulting compound is stable. If specificallynoted, a nitrogen atom in the heterocycle may optionally be quaternized.It is preferred that when the total number of S and O atoms in theheterocycle exceeds one, then these heteroatoms are not adjacent to oneanother. It is preferred that the total number of S and O atoms in theheterocycle is not more than one. Examples of heterocycles include, butare not limited to, 1H-indazole, 2-pyrrolidonyl,2H,6H-1,5,2-dithiazinyl, 2H-pyrrolyl, 3H-indolyl, 4-piperidonyl,4aH-carbazole, 4H-quinolizinyl, 6H-1,2,5-thiadiazinyl, acridinyl,azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl,benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazalonyl,carbazolyl, 4H-carbazolyl, α-, β-, or γ-carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl,isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl,1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, oxazolidinyl., oxazolyl, oxazolidinylpyrimidinyl,phenanthridinyl, phenanthrolinyl, phenoxazinyl, phenazinyl,phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl,piperidinyl, pteridinyl, piperidonyl, 4-piperidonyl, pteridinyl,purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl,pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl,pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl,quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, carbolinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl,thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl,triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl,1,3,4-triazolyl, xanthenyl. Preferred heterocycles include, but are notlimited to, pyridinyl, furanyl, thienyl, pyrrolyl, pyrazolyl,imidazolyl, indolyl, benzimidazolyl, 1H-indazolyl, oxazolidinyl,benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, or isatinoyl.Also included are fused ring and spiro compounds containing, forexample, the above heterocycles.

“Alkoxy” refers to the group R—O— where R is an alkyl group as definedherein.

“Aryloxy” refers to the group R—O— where R is an aryl group, as definedherein.

“Heteroaryloxy” refers to the group R—O— where R is a heteroaryl group,as defined herein.

“Alkanoyl” refers to the group R—C(═O) where R is an alkyl group of 1 to5 carbon atoms.

“Alkanoyloxy” refers to the group R—C(═O)—O where R is an alkyl group of1 to 5 carbon atoms.

“Halo,” refers to chloro, bromo, fluoro, and iodo.

“Haloalkyl,” or “haloaryl” refers to an alkyl or aryl, as defined above,in which one or more hydrogens directly attached to the carbon atoms arereplaced by one or more halo substituents.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances in which it does not. For example, optionally substitutedphenyl indicates either unsubstituted phenyl, or phenyl mono-, di, ortri-substituted, independently, with OH, COOH, lower alkyl, loweralkoxy, halo, nitro, amino, alkylamino, dialkylamino, trifluoromethyland/or cyano.

By “therapeutically effective dose” herein is meant a dose that produceseffects for which it is administered. The exact dose will depend on thepurpose of the treatment, and will be ascertainable by one skilled inthe art using known techniques (see, e.g., Lieberman, PharmaceuticalDosage Forms (Vols. 1-3, 1992); Lloyd, 1999, The Art, Science AndTechnology Of Pharmaceutical Compounding; and Pickar, 1999, DosageCalculations). “Effective amount” refers to an amount of a compound thatcan be therapeutically effective to inhibit, prevent or treat thesymptoms of particular disease, disorder or side effect.

“Pharmaceutically acceptable” refers to those compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem complications commensurate with a reasonablebenefit/risk ratio.

“In combination with”, “combination therapy” and “combination products”refer, in certain embodiments, to the concurrent administration to apatient of a first therapeutic and the compounds as used herein. Whenadministered in combination, each component can be administered at thesame time or sequentially in any order at different points in time.Thus, each component can be administered separately but sufficientlyclosely in time so as to provide the desired therapeutic effect.

“Dosage unit” refers to physically discrete units suited as unitarydosages for the particular individual to be treated. Each unit cancontain a predetermined quantity of active compound(s) calculated toproduce the desired therapeutic effect(s) in association with therequired pharmaceutical carrier. The specification for the dosage unitforms can be dictated by (a) the unique characteristics of the activecompound(s) and the particular therapeutic effect(s) to be achieved, and(b) the limitations inherent in the art of compounding such activecompound(s).

“Stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate,solvate, acid salt hydrate, N-oxide or isomorphic crystalline formthereof” refer to derivatives of the disclosed compounds wherein theparent compound is modified by making acid or base salts thereof.Examples of stereoisomer, prodrug, pharmaceutically acceptable salt,hydrate, solvate, acid salt hydrate, N-oxide or isomorphic crystallineform thereof include, but are not limited to, mineral or organic acidsalts of basic residues such as amines; alkali or organic salts ofacidic residues such as carboxylic acids; and the like. Thestereoisomer, prodrug, pharmaceutically acceptable salt, hydrate,solvate, acid salt hydrate, N-oxide or isomorphic crystalline formthereof include the conventional non-toxic salts or the quaternaryammonium salts of the parent compound formed, for example, fromnon-toxic inorganic or organic acids. For example, such conventionalnon-toxic salts include those derived from inorganic acids such ashydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric andthe like; and the salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,and the like. These physiologically acceptable salts are prepared bymethods known in the art, e.g., by dissolving the free amine bases withan excess of the acid in aqueous alcohol, or neutralizing a freecarboxylic acid with an alkali metal base such as a hydroxide, or withan amine.

Compounds described herein throughout, can be used or prepared inalternate forms. For example, many amino-containing compounds can beused or prepared as an acid addition salt. Often such salts improveisolation and handling properties of the compound. For example,depending on the reagents, reaction conditions and the like, compoundsas described herein can be used or prepared, for example, as theirhydrochloride or tosylate salts. Isomorphic crystalline forms, allchiral and racemic forms, N-oxide, hydrates, solvates, and acid salthydrates, are also contemplated to be within the scope of the presentcompositions and methods.

Certain acidic or basic compounds can exist as zwitterions. All forms ofthe compounds, including free acid, free base and zwitterions, arecontemplated to be within the scope of the present compositions andmethods. It is well known in the art that compounds containing bothamino and carboxyl groups often exist in equilibrium with theirzwitterionic forms. Thus, any of the compounds described hereinthroughout that contain, for example, both amino and carboxyl groups,also include reference to their corresponding zwitterions.

The term “treating” includes the administration of the compounds oragents of the present invention to prevent or delay the onset of thesymptoms, complications, or biochemical indicia of a disease,alleviating the symptoms or arresting or inhibiting further developmentof the disease, condition, or disorder (e.g., cancer). Treatment may beprophylactic (to prevent or delay the onset of the disease, or toprevent the manifestation of clinical or subclinical symptoms thereof)or therapeutic suppression or alleviation of symptoms after themanifestation of the disease.

In general, the phrase “well tolerated” refers to the absence of adversechanges in health status that occur as a result of the treatment andwould affect treatment decisions.

Except when noted, the terms “patient” or “subject” are usedinterchangeably and refer to mammals such as human patients andnon-human primates, as well as experimental animals such as rabbits,rats, and mice, and other animals.

“Prodrug” refers to compounds specifically designed to maximize theamount of active species that reaches the desired site of reaction whichare of themselves typically inactive or minimally active for theactivity desired, but through biotransformation are converted intobiologically active metabolites.

“Stereoisomers” refers to compounds that have identical chemicalconstitution, but differ as regards the arrangement of the atoms orgroups in space.

When any variable occurs more than one time in any constituent or in anyformula, its definition in each occurrence is independent of itsdefinition at every other occurrence. Combinations of substituentsand/or variables are permissible only if such combinations result instable compounds.

“Short chain dehydrogenase reductase (SDR)” refers to a family ofNAD(P)(H) dependent oxido-reductases represent a diverse family ofgreater than 63 human proteins. These enzymes are responsible for theoxidation or reduction of a wide range of endogenous (prostaglandins,steroid hormones, retinal, dihydropteridin, UDP, and trans 2-enoyl CoA)and exogenous chemicals (e.g., anthracyclin drugs).

“Modulate” refers to the suppression, enhancement or induction of afunction or condition. For example, the pyrazolo pyrimidine or AB-129compounds, derivatives and analogs thereof of the invention can modulatecancer by inhibition of short chain dehydrogenase reductase (SDR) enzymeactivity. For example, AB-129 compounds, derivatives and analogs thereofcan inhibit carbonyl reductase 1 (CBR1) activity in lung carcinoma cellsthereby alleviating lung cancer by inhibiting or reducing growth of lungcarcinoma cells.

“Carbonyl reductase” refers to a family of enzymes, for example,carbonyl reductase 1 (NADPH: secondary-alcohol oxidoreductase) which ispart of a group of NADPH-dependent cytosolic enzymes called short chaindehydrogenase/reductase (SDR) that catalyze the reduction of variouscarbonyl compounds to their corresponding alcohols. The enzyme isubiquitous in nature and acts on a large number of biologically andpharmacologically active compounds. Carbonyl reductase is believed tofunction physiologically as dehydrogenases of prostaglandins orhydroxysteroids, as well as in drug metabolism.

“11β-hydroxysteroid dehydrogenase (11β-HSD)” refers to11β-hydroxysteroid dehydrogenase I or 11β-hydroxysteroid dehydrogenaseII, or an enzyme with a related activity.

“17β-hydroxysteroid dehydrogenase(17β-HSD)” refers to 17β-hydroxysteroiddehydrogenase 1 or 17β-hydroxysteroid dehydrogenase II, or an enzymewith a related activity.

“Anthracycline anti-cancer agent” refers to the family of anthracyclineantibiotic drugs that include, for example daunorubicin, doxorubicin,epirubicin, and idarubicin.

“Cardiotoxic side effect” refers to acute or chronic cardiomyopathy thatcan lead to congestive heart failure. The development of chroniccardiotoxicity is clinically important. Chronic cardiotoxicity candevelop many years after treatment with anthracyclines. Children andyounger adults treated with anthracyclines are exposed to a lifetimerisk of developing serious cardiomyopathy. Because cancer patients arenot usually monitored for more than 5-7 years, the number of thesepatients developing late-onset cardiomyopathies can be expected toincrease substantially in the future.

“Cancer” or “malignancy” are used as synonymous terms and refer to anyof a number of diseases that are characterized by uncontrolled, abnormalproliferation of cells, the ability of affected cells to spread locallyor through the bloodstream and lymphatic system to other parts of thebody (i.e., metastasize) as well as any of a number of characteristicstructural and/or molecular features. A “cancerous” or “malignant cell”is understood as a cell having specific structural properties, lackingdifferentiation and being capable of invasion and metastasis. Examplesof cancers are kidney, colon, breast, prostate and liver cancer. (seeDeVita, V. et al. (eds.), 2001, CANCER PRINCIPLES AND PRACTICE OFONCOLOGY, 6th. Ed., Lippincott Williams & Wilkins, Philadelphia, Pa.;this reference is herein incorporated by reference in its entirety forall purposes).

“Cancer-associated” refers to the relationship of a nucleic acids andits expression, or lack thereof, or a protein and its level or activity,or lack thereof, to the onset of malignancy in a subject cell. Forexample, cancer can be associated with expression of a particular genethat is not expressed, or is expressed at a lower level, in a normalhealthy cell. Conversely, a cancer-associated gene can be one that isnot expressed in a malignant cell (or in a cell undergoingtransformation), or is expressed at a lower level in the malignant cellthan it is expressed in a normal healthy cell.

“Neoplastic cells” and “neoplasia” refer to cells which exhibitrelatively autonomous growth, so that they exhibit an aberrant growthphenotype characterized by a significant loss of control of cellproliferation. Neoplastic cells comprise cells which can be activelyreplicating or in a temporary non-replicative resting state (G1 or G0);similarly, neoplastic cells can comprise cells which have awell-differentiated phenotype, a poorly-differentiated phenotype, or amixture of both type of cells. Thus, not all neoplastic cells arenecessarily replicating cells at a given timepoint. The set defined asneoplastic cells consists of cells in benign neoplasms and cells inmalignant (or frank) neoplasms. Frankly neoplastic cells are frequentlyreferred to as cancer (discussed supra), typically termed carcinoma iforiginating from cells of endodermal or ectodermal histological origin,or sarcoma if originating from cell types derived from mesoderm.

In the context of the invention, the term “transformation” refers to thechange that a normal cell undergoes as it becomes malignant. Ineukaryotes, the term “transformation” can be used to describe theconversion of normal cells to malignant cells in cell culture.

“Proliferating cells” are those which are actively undergoing celldivision and growing exponentially.

“Loss of cell proliferation control” refers to the property of cellsthat have lost the cell cycle controls that normally ensure appropriaterestriction of cell division. Cells that have lost such controlsproliferate at a faster than normal rate, without stimulatory signals,and do not respond to inhibitory signals.

“Leukemia” refers to cancer of cells in the bloodstream or lymphaticsystem. Types of leukemia include but are not limited to, AcuteLymphoblastic Leukemia (Adult or Childhood), Acute Myeloid Leukemia(Adult or Childhood), Chronic Lymphocytic Leukemia, Chronic MyelogenousLeukemia, or Hairy Cell Leukemia.

“Kaposi's sarcoma (KS)” refers to a sarcoma that develops in connectivetissues such as cartilage, bone, fat, muscle, blood vessels, or fibroustissues (related to tendons or ligaments). The vast majority of KS caseshave developed in association with human immunodeficiency virus (HIV)infection and the acquired immunodeficiency syndrome (AIDS). KS tumorsdevelop in the tissues below the skin surface, or in the mucousmembranes of the mouth, nose, or anus.

“Inflammation” or “inflammatory response” refers to an innate immuneresponse that occurs when tissues are injured by bacteria, trauma,toxins, heat, or any other cause. The damaged tissue releases compoundsincluding histamine, bradykinin, and serotonin. Inflammation refers toboth acute responses (i.e., responses in which the inflammatoryprocesses are active) and chronic responses (i.e., responses marked byslow progression and formation of new connective tissue). Acute andchronic inflammation can be distinguished by the cell types involved.Acute inflammation often involves polymorphonuclear neutrophils; whereaschronic inflammation is normally characterized by a lymphohistiocyticand/or granulomatous response. Inflammation includes reactions of boththe specific and non-specific defense systems. A specific defense systemreaction is a specific immune system reaction response to an antigen(possibly including an autoantigen). A non-specific defense systemreaction is an inflammatory response mediated by leukocytes incapable ofimmunological memory. Such cells include granulocytes, macrophages,neutrophils and eosinophils. Examples of specific types of inflammationare diffuse inflammation, focal inflammation, croupous inflammation,interstitial inflammation, obliterative inflammation, parenchymatousinflammation, reactive inflammation, specific inflammation, toxicinflammation and traumatic inflammation.

“Diabetes mellitus” or “diabetes” refers to a disease or condition thatis generally characterized by metabolic defects in production andutilization of glucose which result in the failure to maintainappropriate blood sugar levels in the body. The result of these defectsis elevated blood glucose, referred to as “hyperglycemia.” Two majorforms of diabetes are Type 1 diabetes and Type 2 diabetes. As describedabove, Type 1 diabetes is generally the result of an absolute deficiencyof insulin, the hormone which regulates glucose utilization. Type 2diabetes often occurs in the face of normal, or even elevated levels ofinsulin and can result from the inability of tissues to respondappropriately to insulin. Most Type 2 diabetic patients are insulinresistant and have a relative deficiency of insulin, in that insulinsecretion can not compensate for the resistance of peripheral tissues torespond to insulin. In addition, many Type 2 diabetics are obese. Othertypes of disorders of glucose homeostasis include Impaired GlucoseTolerance, which is a metabolic stage intermediate between normalglucose homeostasis and diabetes, and Gestational Diabetes Mellitus,which is glucose intolerance in pregnancy in women with no previoushistory of Type 1 or Type 2 diabetes.

“Secondary diabetes” is diabetes resulting from other identifiableetiologies which include: genetic defects of β cell function (e.g.,maturity onset-type diabetes of youth, referred to as “MODY,” which isan early-onset form of Type 2 diabetes with autosomal inheritance; see,e.g., Fajans S. et al., Diabet. Med. 9 Suppl 6: S90-5, 1996, and Bell,G. et al., Annu. Rev. Physiol. 58: 171-86, 1996); genetic defects ininsulin action; diseases of the exocrine pancreas (e.g.,hemochromatosis, pancreatitis, and cystic fibrosis); certain endocrinediseases in which excess hormones interfere with insulin action (e.g.,growth hormone in acromegaly and cortisol in Cushing's syndrome);certain drugs that suppress insulin secretion (e.g., phenyloin) orinhibit insulin action (e.g., estrogens and glucocorticoids); anddiabetes caused by infection (e.g., rubella, Coxsackie, and CMV); aswell as other genetic syndromes.

The guidelines for diagnosis for Type 2 diabetes, impaired glucosetolerance, and gestational diabetes have been outlined by the AmericanDiabetes Association (see, e.g., The Expert Committee on the Diagnosisand Classification of Diabetes Mellitus, Diabetes Care, 2 (Suppl 1):S5-19, 1999).

Methods of Treatment

Short chain dehydrogenases/reductases (SDRs), for example, carbonylreductase 1 (CBR1) have a role in metabolism and disease. AB129-typecompounds and analogs thereof are inhibitors of the SDR enzyme family.AB129-type compounds and analogs are useful for medical treatment, forexample, cancer therapy, and have been shown to have biologicalactivity.

(1) Human carbonyl reductase 1 (CBR1) is within the family of shortchain dehydrogenases/reductases (SDRs).

(2) The SDR enzyme family has more than 1600 members. Greater than 63SDR enzymes are found in human.

(3) SDR enzymes catalyze an NAD(P)(H)-dependent oxidoreduction ordehydrogenation.

(4) The catalytic active site of the SDR enzyme comprises an S/YxxxKcatalytic triad.

(5) The function of SDR enzymes include intermediary metabolism, lipidhormone metabolism (e.g., steroids, prostaglandins, retinols/retinals)and enzymes of unknown function.

(6) SDR enzymes are correlated with many genetic and metabolicdisorders.

(7) SDR enzymes can regulate the nuclear hormone switch (e.g.,cortisone, estradiol, prostaglandin) as an important regulatory targetby AB129-type compounds.

(8) AB129-type compounds and analogs thereof that inhibit carbonylreductase 1 (CBR1) activity are useful for treatment of lung cancer,colon cancer, metastatic cancer, or cancer drug resistance.

(9) AB129-type compounds and analogs thereof that inhibit11β-hydroxysteroid dehydrogenase activity and result in decreased levelsof cortisone are useful for treatment of diabetes or obesity.

(10) AB129-type compounds and analogs thereof that inhibit17β-hydroxysteroid dehydrogenase activity are useful for treatment ofinflammatory disease, ovarian cancer or breast cancer.

In one embodiment, the present invention is directed to a compound ofFormula I or II:

or a pharmaceutically-acceptable salt or prodrug thereof;

wherein:

-   -   Y is N or CR₅;    -   Z is NR₃R₄, halo, H, OH, alkyl, alkyloxy, or haloalkyl;    -   R_(1a) is indolyl, thiazolyl, benzyl, biphenylyl, thiophenyl,        pyrrolyl, or phenyl, wherein said phenyl is substituted with at        least one of OH, —NR₃R₄,—C(═O)NR₆R₇, —CN, NO₂—C(═O)OH,        —C(═O)O-alkyl, (C₁-C₄)alkyl, halo, haloalkyl or haloaryl; and        wherein said indolyl, thiazolyl, benzyl, biphenylyl, thiophenyl,        or pyrrolyl is optionally substituted with OH, —NR₃R₄,        —C(═O)NR₆R₇, —CN, NO₂, —C(═O)O—R₃, (C₁-C₄)alkyl, halo, haloalkyl        or haloaryl;    -   R_(1b) is indolyl, thiazolyl, benzyl, biphenylyl, thiophenyl,        pyrrolyl, or phenyl wherein said indoyl, thiazolyl, benzyl,        biphenylyl, thiophenyl, pyrrolyl, phenyl is optionally        substituted with —OH, —NR₃R₄, —C(═O)NR₆R₇, —CN, NO₂, —C(═O)O—R₃,        (C₁-C₄)alkyl, halo, haloalkyl, or haloaryl;    -   R₂ is C₁-C₆ alkyl or C₄-C₇ cycloalkyl, wherein said alkyl or        said cycloalkyl is optionally substituted with mono- or        di-alkoxy, mono- or di-halophenyl, mono- or di-(C₁₋₄)alkoxy        phenyl, mono- or di-(C₁₋₄)alkyl phenyl, perhalo(C₁₋₄)alkyl        phenyl, carboxyl, tert-butyl carboxyl, phosphoryl, (C₁₋₆)alkyl,        (C₄₋₇)cycloalkyl, indolyl, isoindolyl, pyridyl, naphthyl,        pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl,        pyrazinyl, pyridazinyl, furyl, thienyl, or alkylmorpholino;    -   R₃ and R₄ are independently H, C₁-C₆ alkyl, t-Boc,        morpholino(C₁-C₄)alkyl, carboxy(C₁-C₃)alkyl,        (C₁-C₄)alkoxycarbonyl(C₁-C₃)alkyl, aryl, heteroaryl, aryloxy,        heterocycle, cycloalkyl, alkenyl with the proviso that the        double bond of the alkenyl is not present at the carbon atom        that is directly linked to N, alkynyl with the proviso that the        triple bond of the alkynyl is not present at the carbon atom        that is directly linked to N, perfluoroalkyl, —S(O)₂alkyl,        —S(O)₂aryl, —(C═O)heteroaryl, —(C═O)aryl, —(C═O)(C₁-C₆) alkyl,        —(C═O)cycloalkyl, —(C═O)heterocycle, alkyl-heterocycle, aralkyl,        arylalkenyl, —CONR₆R₇, —SO₂R₆R₇, arylalkoxyalkyl,        arylalkylalkoxy, heteroarylalkylalkoxy, aryloxyalkyl,        heteroaryloxyalkyl, aryloxyaryl, aryloxyheteroaryl,        alkylaryloxyaryl, alkylaryloxyheteroaryl, alkylaryloxyalkyamine,        alkoxycarbonyl, aryloxycarbonyl, or heteroaryloxycarbonyl;    -   R₅ are independently H, —OH, halo, optionally monosubstituted        (C₁-C₆)alkyl, optionally monosubstituted (C₁-C₄)alkoxycarbonyl,        optionally monosubstituted (C₁-C₄)alkanoyl, carbamoyl,        optionally monosubstituted (C₁-C₄)alkyl carbamoyl, phenyl,        halophenyl, optionally monosubstituted (C₁-C₄)alkylphenyl,        optionally monosubstituted (C₁-C₄)alkoxyphenyl, or optionally        monosubstituted perhalo(C₁-C₄)alkylphenyl, wherein said optional        substitution is (C₁-C₄)alkyl, OH, or halogen;    -   R₆ and R₇ are independently H, alkyl, aryl, heteroaryl,        alkylaryl, arylalkyl, heteroarylalkyl, or alkylheteroaryl;    -   provided the compound is not        1-tert-butyl-3-p-tolyl-1H-pyrazolo[3,4-c]pyrimidin-4-ylamine.

In certain embodiments, Y is N.

In a detailed embodiment, R_(1a) or R_(1b) is phenyl substituted withmono, di or tri-OH. In a further detailed embodiment, the phenyl isfurther substituted with a halo. In a further detailed embodiment, thehalo is F.

In a detailed embodiment, R₂ is 2-methyl-propane. In a detailedembodiment, R₃ and R₄ are H. In a detailed embodiment, R₅ is H. In adetailed embodiment, R₆ is H and R₇ is methyl.

In certain embodiments, R_(1a) is, independently, phenyl substituted ata meta position with —CH₃, tert-butyl, —CF₃ or halo. In a detailedembodiment, R_(1a) is, independently, phenyl substituted at a metaposition with halo, alkyl, haloalkyl, haloaryl, aryl, O-alkyl, CN, NO₂,CO—O—R₃, CO—N(R₃)₂. In a detailed embodiment, Z is F, Br Cl, or I

In a detailed embodiment, the compounds of formula I or formula IIinclude:

-   3-(4-amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-phenol;-   3-(7-isopropyl-4-methylamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-phenol;-   [5-(3-amino-phenyl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-methyl-amine;-   3-(4-benzylamino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-phenol;-   3-(4-dibenzylamino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-phenol;-   3-[5-(3-hydroxy-phenyl)-4-methylamino-pyrrolo[2,3-d]pyrimidin-7-yl]-propionic    acid tert-butyl ester;-   3-[5-(3-hydroxy-phenyl)-4-methylamino-pyrrolo[2,3-d]pyrimidin-7-yl]-propionic    acid;-   3-bromo-5-(7-isopropyl-4-methylamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-phenol;-   3-(7-isopropyl-4-methylamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-5-methyl-phenol;-   3-tert-Butyl-5-(7-isopropyl-4-methylamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-phenol;-   3-(7-Isopropyl-4-methylamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-5-trifluoromethyl-phenol;-   3-bromo-5-(4-chloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-phenol;-   3-(4-chloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-5-methyl-phenol;-   3-tert-butyl-5-(4-chloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-phenol;-   3-(4-Chloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-5-trifluoromethyl-phenol

or a pharmaceutically-acceptable salt or prodrug thereof.

In a further detailed embodiment, the compound has the formula:

In a further detailed embodiment, the compound has the formula:

In a further detailed embodiment, the compound has the formula:

In a further detailed embodiment, the compound has the formula:

In a further detailed embodiment, the compound has the formula:

In another embodiment, the pharmaceutical composition, comprises apharmaceutically acceptable carrier, and the compound. In a detailedembodiment the pharmaceutical composition further comprises at least oneanthracycline compound, including but not limited to, daunorubicindoxorubicin, epirubicin, idarubicin, or a mixture thereof.

In another embodiment, methods for preventing or treating a disease orcondition associated with carbonyl reductase I in a mammalian areprovided, comprising the step of administering to the mammal acomposition comprising an effective amount of the compound.

In a further embodiment, the disease state is cancer. In a detailedembodiment, the cancer is lung cancer.

In another embodiment, methods for identifying a therapeutic cancertreatment are provided, comprising contacting a tumor cell culture withan effective amount of the compound.

In another embodiment, methods for alleviating a disease state in amammal believed to be responsive to treatment with an inhibitor ofcarbonyl reductase 1 are provided, comprising administering to themammal an effective amount of the compound, in combination with aneffective amount of an anthracycline anti-cancer agent, wherein thedisease state of the mammal is alleviated. In a detailed embodiment, theanthracycline anti-cancer agent includes, but is not limited to,daunorubicin, doxorubicin, epirubicin, or idarubicin. In a furtherdetailed embodiment, the potency of the anthracycline anti-cancer agentis maintained in the absence of a cardiotoxic side effect. In a detailedembodiment, the disease state is cancer. In a further detailedembodiment, the disease state is selected from cancer, metastaticcancer, colon cancer, ovarian cancer, leukemia, lymphoma, myeloma, acutemyeloid leukemia, acute lymphocytic leukemia, chronic myelogenousleukemia, neuroblastoma, lung cancer, breast cancer, acquiredimmunodeficiency syndrome (AIDS)-associated Kaposi's sarcoma(KS),inflammation, obesity, or diabetes.

In another embodiment, methods of preventing or treating a disease orcondition associated with the synthesis of prostaglandin E in a mammalcomprises administering to the mammal a effective amount of the compoundwherein the disease state of the mammal is alleviated. In a detailedembodiment, the disease state is metastatic cancer. In a detailedembodiment, the disease state is colon cancer.

In another embodiment, methods for alleviating a disease state in amammal believed to be responsive to treatment with an inhibitor of shortchain dehydrogenase/reductase (SDR) family of NAD(P)(H) dependentoxido-reductases, comprise administering to the mammal a effectiveamount of the compound wherein the disease state of the mammal isalleviated. In a detailed embodiment, the therapeutic amount of thecompound inhibits 11β-hydroxysteroid dehydrogenase I. In a detailedembodiment, the therapeutic amount of the compound inhibits11β-hydroxysteroid dehydrogenase II.

In a detailed embodiment, the therapeutic amount of the compoundstimulates synthesis of cortisol. In a further detailed embodiment, thedisease state is inflammation.

In a detailed embodiment, the therapeutic amount of the compoundstimulates degradation of cortisone. In a further detailed embodiment,the therapeutic amount of the compound alleviates the disease stateselected from obesity or diabetes.

In a detailed embodiment, the therapeutic amount of the compoundinhibits 17β-hydroxysteroid dehydrogenases. In a further detailedembodiment, the therapeutic amount of the compound alleviates thedisease state selected from inflammation, ovarian cancer or breastcancer.

In another embodiment, methods for identifying a therapeutic cancertreatment are provided comprising the steps of: contacting a tumor cellculture with an effective amount of a according to claim 1; measuringgrowth inhibition of the tumor cells in culture; and identifying atherapeutic cancer treatment for a mammalian subject by inhibition ofthe tumor cell growth in culture

In another embodiment, methods for preventing or treating cancer in amammal are provided comprising the step of administering to the mammalan effective amount of the compound. In a detailed embodiment, thecancer is lung cancer, metastatic cancer, colon cancer, ovarian cancer,leukemia, lymphoma, myeloma, acute myeloid leukemia, acute lymphocyticleukemia, chronic myelogenous leukemia, neuroblastoma, breast cancer,acquired immunodeficiency syndrome (AIDS)-associated Kaposi's sarcoma(KS).

Pharmaceutical Compositions

Inhibitors and modulators of SDR type enzymes, for example, AB129-typecompounds and analogs thereof, are useful in the present compositionsand methods and can be administered to a human patient per se, in theform of a stereoisomer, prodrug, pharmaceutically acceptable salt,hydrate, solvate, acid salt hydrate, N-oxide, prodrug ester, orisomorphic crystalline form thereof, or in the form of a pharmaceuticalcomposition where the compound is mixed with suitable carriers orexcipient(s) in a therapeutically effective amount, for example, lungcancer or colon cancer. “Prodrug esters” as employed herein includesprodrug esters which are known in the art for carboxylic and phosphorusacid esters such as methyl, ethyl, benzyl and the like.

Routes of Administration

Pharmaceutical compositions of inhibitors and modulators of SDR typeenzymes, for example, AB129-type compounds and analogs thereof,described herein can be administered by a variety of routes. Suitableroutes of administration can, for example, include oral, rectal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, spinal, epidural, intranasal, or intraocularinjections. Alternatively, one can administer the compound in a localrather than systemic manner, for example via injection of the compounddirectly into the subject, often in a depot or sustained releaseformulation. Furthermore, one can administer the compound in a targeteddrug delivery system, for example, in a liposome coated vesicle. Theliposomes can be targeted to and taken up selectively by the tissue ofchoice. In a further embodiment, the pharmaceutical compositions ofAB129-type compounds and analogs described herein are administeredorally.

Composition/Formulation

The pharmaceutical compositions described herein can be manufactured ina manner that is itself known, e.g., by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes. Pharmaceuticalcompositions for use as described herein can be formulated inconventional manner using one or more physiologically acceptablecarriers comprising excipients and auxiliaries which facilitateprocessing of the active compounds into preparations which can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen. For injection, the agents can be formulated inaqueous solutions, e.g., in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art. For oral administration, the compounds can beformulated readily by combining with pharmaceutically acceptablecarriers that are well known in the art. Such carriers enable thecompounds to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained by mixing the compounds with a solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable auxiliaries, if desired, to obtaintablets or dragee cores.

Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents can beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions can be used, which can optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments can be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds can be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers can be added. All formulations fororal administration should be in dosages suitable for suchadministration. For buccal administration, the compositions can take theform of tablets or lozenges formulated in conventional manner. Foradministration by inhalation, the compounds for use are convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator can be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection can be presented in unit dosage form, e.g., in ampules orin multi-dose containers, with an added preservative. The compositionscan take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Pharmaceutical formulations forparenteral administration include aqueous solutions of the activecompounds in water-soluble form. Additionally, suspensions of the activecompounds can be prepared as appropriate oily injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides, or liposomes. Aqueous injection suspensions can containsubstances which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, thesuspension can also contain suitable stabilizers or agents whichincrease the solubility of the compounds to allow for the preparation ofhighly concentrated solutions. Alternatively, the active ingredient canbe in powder form for constitution with a suitable vehicle, e.g.,sterile pyrogen-free water, before use.

The compounds can also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides. In additionto the formulations described previously, the compounds can also beformulated as a depot preparation. Such long acting formulations can beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds can be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

A suitable pharmaceutical carrier for hydrophobic compounds is acosolvent system comprising benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. The cosolventsystem can be the VPD co-solvent system. VPD is a solution of 3% (w/v)benzyl alcohol, 8% (w/v) of the nonpolar surfactant polysorbate 80, and65% (w/v) polyethylene glycol 300, made up to volume in absoluteethanol. The VPD co-solvent system (VPD:SW) consists of VPD diluted 1:1with a 5% (w/v) dextrose in water solution. This co-solvent systemdissolves hydrophobic compounds well, and itself produces low toxicityupon systemic administration. Naturally, the proportions of a co-solventsystem can be varied considerably without destroying its solubility andtoxicity characteristics. Furthermore, the identity of the co-solventcomponents can be varied: for example, other low-toxicity nonpolarsurfactants can be used instead of polysorbate 80; the fraction size ofpolyethylene glycol can be varied; other biocompatible polymers canreplace polyethylene glycol, e.g. polyvinyl pyrrolidone; and othersugars or polysaccharides can substitute for dextrose. Alternatively,other delivery systems for hydrophobic pharmaceutical compounds can beemployed. Liposomes and emulsions are well known examples of deliveryvehicles or carriers for hydrophobic drugs. Certain organic solventssuch as dimethylsulfoxide also can be employed, although usually at thecost of greater toxicity.

Additionally, the compounds can be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various types of sustained-releasematerials have been established and are well known by those skilled inthe art. Sustained-release capsules can, depending on their chemicalnature, release the compounds for a few weeks up to over 100 days. Thepharmaceutical compositions also can comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions foradministering the AB129 (see, e.g., Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa. 18^(th) ed., 1990, incorporated hereinby reference). The pharmaceutical compositions generally comprise adifferentially expressed protein, agonist or antagonist in a formsuitable for administration to a patient. The pharmaceuticalcompositions are generally formulated as sterile, substantially isotonicand in full compliance with all Good Manufacturing Practice (GMP)regulations of the U.S. Food and Drug Administration.

Effective Dosages

Pharmaceutical compositions suitable for use include compositionswherein the AB129-type compounds and analogs are contained in atherapeutically effective amount. Determination of an effective amountis well within the capability of those skilled in the art, especially inlight of the detailed disclosure provided herein. For any compound usedin the present method, a therapeutically effective dose can be estimatedinitially from cell culture assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅₀ as determined in cell culture (i.e., theconcentration of test compound that is lethal to 50% of a cell culture)or the IC₅₀ as determined in cell culture (i.e., the concentration ofcompound that is lethal to 100% of a cell culture). Such information canbe used to more accurately determine useful doses in humans. Initialdosages can also be formulated by comparing the effectiveness of theAB129-type compounds and analogs described herein in cell culture assayswith the effectiveness of known cancer treatments. In this method aninitial dosage can be obtained by multiplying the ratio of effectiveconcentrations obtained in cell culture assay for the AB129-typecompounds and analogs and a known cancer treatment by the effectivedosage of the known cancer treatment. For example, if an AB129-typecompound or analog is twice as effective in cell culture assay than thecancer treatment (i.e., the IC₅₀ of AB129 is equal to one half times theIC₅₀ cancer treatment in the same assay), an initial effective dosage ofthe AB129-type compound or analog would be one-half the known dosage forthe cancer treatment. Using these initial guidelines one having ordinaryskill in the art could determine an effective dosage in humans. Initialdosages can also be estimated from in vivo data. One having ordinaryskill in the art could readily optimize administration to humans basedon this data. Dosage amount and interval can be adjusted individually toprovide plasma levels of the active compound which are sufficient tomaintain therapeutic effect. Usual patient dosages for oraladministration range from about 50-2000 mg/kg/day, typically from about250-1000 mg/kg/day, from about 500-700 mg/kg/day or from about 350-550mg/kg/day. Therapeutically effective serum levels will be achieved byadministering multiple doses each day. In cases of local administrationor selective uptake, the effective local concentration of the drug cannot be related to plasma concentration. One having skill in the art willbe able to optimize therapeutically effective local dosages withoutundue experimentation. The amount of composition administered will, ofcourse, be dependent on the subject being treated, on the subject'sweight, the severity of the affliction, the manner of administration andthe judgment of the prescribing physician. The therapy can be repeatedintermittently while lung cancer or colon cancer is detectable or evenwhen they are not detectable. Moreover, due to its apparent nontoxicity,the therapy can be provided alone or in combination with other drugs,such as for example, anti-inflammatories, antibiotics, corticosteroids,vitamins and the like. Possible synergism between the AB129-typecompounds or analogs described herein and other drugs can occur. Inaddition, possible synergism between a plurality of AB129-type compoundsor analogs can occur.

The typical daily dose of a pharmaceutical composition of inhibitors andmodulators of SDR type enzymes, for example, AB129-type compounds andanalogs thereof, varies according to individual needs, the condition tobe treated and with the route of administration. Suitable doses are inthe general range of from 0.001 to 10 mg/kg bodyweight of the recipientper day. Within this general dosage range, doses can be chosen at whichthe pharmaceutical composition of AB129-type compounds and analogs has apositive effect on cancer treatment efficacy. In general, but notexclusively, such doses will be in the range of from 0.5 to 10 mg/kg.

In addition, within the general dose range, doses can be chosen at whichthe compounds pharmaceutical composition of AB129-type compounds andanalogs has a positive effect on cancer treatment efficacy. In general,but not exclusively, such doses will be in the range of from 0.001 to0.5 mg/kg. It is to be understood that the 2 sub ranges noted above arenot mutually exclusive and that the particular activity encountered at aparticular dose will depend on the nature of the pharmaceuticalcomposition of AB129-type compounds and analogs used.

The pharmaceutical composition of AB129-type compounds and analogs canbe in unit dosage form, for example, a tablet or a capsule so that thepatient can self-administer a single dose. In general, unit dosescontain in the range of from 0.05-100 mg of a compound of thepharmaceutical composition of AB129-type compounds and analogs. Unitdoses contain from 0.05 to 10 mg of the pharmaceutical composition. Theactive ingredient can be administered from 1 to 6 times a day. Thusdaily doses are in general in the range of from 0.05 to 600 mg per day.In an embodiment, daily doses are in the range of from 0.05 to 100 mgper day or from 0.05 to 5 mg per day.

Toxicity

Toxicity and therapeutic efficacy of inhibitors and modulators of SDRtype enzymes, for example, AB129-type compounds and analogs thereof,described herein can be determined by standard pharmaceutical proceduresin cell cultures or experimental animals, e.g., by determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index and can beexpressed as the ratio between LD₅₀ and ED₅₀ Compounds which exhibithigh therapeutic indices are chosen. The data obtained from these cellculture assays and animal studies can be used in formulating a dosagerange that is not toxic for use in human. The dosage of such compoundslies within a range of circulating concentrations that include theED_(5o) with little or no toxicity. The dosage can vary within thisrange depending upon the dosage form employed and the route ofadministration utilized. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition. (See, e.g., Fingl et al., 1975, In: ThePharmacological Basis of Therapeutics, Ch. 1, p. 1). One of theadvantages, among others, of using the AB129-type compounds and analogsdescribed herein to treat disease, e.g., lung cancer or colon cancer istheir lack of toxicity. For example, it has been found that repeatedintraperitoneal doses of 75 mg/kg produced no ill effects in mice (seeExample 5). Since the i.v. serum half-life (t_(1/2)) of AB129 is about2-2.5 hours, repeated daily dosages of the AB129 described hereinwithout ill effects is predictable.

Diagnostic Methods

In addition to assays, the creation of animal models, and nucleic acidbased therepeutics, identification of important differentially expressedgenes allows the use of these genes in diagnosis (e.g., diagnosis ofcell states and abnormal epithelial cell conditions). Disorders based onmutant or variant differentially expressed genes can be determined.Methods for identifying cells containing variant differentiallyexpressed genes comprising determining all or part of the sequence of atleast one endogenous differentially expressed genes in a cell areprovided. As will be appreciated by those in the art, this can be doneusing any number of sequencing techniques. Methods of identifying thedifferentially expressed genotype of an individual comprisingdetermining all or part of the sequence of at least one differentiallyexpressed gene of the individual are also provided. This is generallydone in at least one tissue of the individual, and can include theevaluation of a number of tissues or different samples of the sametissue. The method can include comparing the sequence of the sequenceddifferentially expressed gene to a known differentially expressed gene,i.e., a wild-type gene.

The sequence of all or part of the differentially expressed gene canthen be compared to the sequence of a known differentially expressedgene to determine if any differences exist. This can be done using anynumber of known sequence identity programs, such as Bestfit, and othersoutlined herein. In some methods, the presence of a difference in thesequence between the differentially expressed gene of the patient andthe known differentially expressed gene is indicative of a disease stateor a propensity for a disease state, as outlined herein.

Similarly, diagnosis of epithelial cell states can be done using themethods and compositions herein. By evaluating the gene expressionprofile of epithelial cells from a patient, the epithelial cell statecan be determined. This is particularly useful to verify the action of adrug, for example an immunosuppressive drug. Other methods compriseadministering the drug to a patient and removing a cell sample,particularly of epithelial cells, from the patient. The gene expressionprofile of the cell is then evaluated, as outlined herein, for exampleby comparing it to the expression profile from an equivalent sample froma healthy individual. In this manner, both the efficacy (i.e., whetherthe correct expression profile is being generated from the drug) and thedose (is the dosage correct to result in the correct expression profile)can be verified.

The present discovery relating to the role of differentially expressedin epithelial cells thus provides methods for inducing or maintainingdiffering epithelial cell states. In one method, the differentiallyexpressed proteins, and particularly differentially expressed fragments,are useful in the study or treatment of conditions which are mediated byepithelial cell activity, i.e., to diagnose, treat or prevent epithelialcell-mediated disorders. Thus, “epithelial cell mediated disorders” or“disease states” can include conditions involving, for example,arthritis, diabetes, or multiple sclerosis.

Methods of modulating epithelial cell activity in cells or organisms areprovided. Some methods comprise administering to a cell ananti-differentially expressed antibody or other agent identified hereinor by the methods provided herein, that reduces or eliminates thebiological activity of the endogenous differentially expressed protein.Alternatively, the methods comprise administering to a cell or organisma recombinant nucleic acid encoding a differentially expressed proteinor modulator including anti-sense nucleic acids. As will be appreciatedby those in the art, this can be accomplished in any number of ways. Insome methods, the activity of differentially expressed is increased byincreasing the amount of differentially expressed in the cell, forexample by overexpressing the endogeneous differentially expressed or byadministering a differentially expressed gene, using known gene therapytechniques, for example. In one method, the gene therapy techniquesinclude the incorporation of the exogenous gene using enhancedhomologous recombination (EHR), for example as described inPCT/US93/03868, hereby incorporated by reference in its entirety.

Methods for diagnosing an epithelial cell activity related condition inan individual are provided. The methods comprise measuring the activityof differentially expressed protein in a tissue from the individual orpatient, which can include a measurement of the amount or specificactivity of the protein. This activity is compared to the activity ofdifferentially expressed from either an unaffected second individual orfrom an unaffected tissue from the first individual. When theseactivities are different, the first individual can be at risk for anepithelial cell activity mediated disorder.

Furthermore, nucleotide sequences encoding a differentially expressedprotein can also be used to construct hybridization probes for mappingthe gene which encodes that differentially expressed protein and for thegenetic analysis of individuals with genetic disorders. The nucleotidesequences provided herein can be mapped to a chromosome and specificregions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

Kits

The differentially expressed protein, agonist or antagonist or theirhomologs are useful tools for examining expression and regulation ofsignaling in epithelial cells via the PAR1 pathway. Reagents thatspecifically hybridize to nucleic acids encoding differentiallyexpressed proteins (including probes and primers of the differentiallyexpressed proteins), and reagents that specifically bind to thedifferentially expressed proteins, e.g., antibodies, are used to examineexpression and regulation.

Nucleic acid assays for the presence of differentially expressedproteins in a sample include numerous techniques are known to thoseskilled in the art, such as Southern analysis, northern analysis, dotblots, RNase protection, 51 analysis, amplification techniques such asPCR and LCR, high density oligonucleotide array analysis, and in situhybridization. In in situ hybridization, for example, the target nucleicacid is liberated from its cellular surroundings in such as to beavailable for hybridization within the cell while preserving thecellular morphology for subsequent interpretation and analysis. Thefollowing articles provide an overview of the art of in situhybridization: Singer et al., Biotechniques 4: 230-250, 1986; Haase etal., Methods in Virology, vol. VII, pp. 189-226, 1984; and Nucleic AcidHybridization: A Practical Approach (Hames et al., eds. 1987), eachincorporated herein by reference. In addition, a differentiallyexpressed protein can be detected with the various immunoassaytechniques described above. The test sample is typically compared toboth a positive control (e.g., a sample expressing recombinantdifferentially expressed protein) and a negative control.

Kits for screening epithelial cell activity modulators. Such kits can beprepared from readily available materials and reagents are provided. Forexample, such kits can comprise any one or more of the followingmaterials: the differentially expressed proteins, agonists, orantagonists, reaction tubes, and instructions for testing the activitiesof differentially expressed genes. A wide variety of kits and componentscan be prepared depending upon the intended user of the kit and theparticular needs of the user. For example, the kit can be tailored forin vitro or in vivo assays for measuring the activity of adifferentially expressed proteins or epithelial cell activitymodulators.

Kits comprising probe arrays as described above are provided. Optionaladditional components of the kit include, for example, other restrictionenzymes, reverse-transcriptase or polymerase, the substrate nucleosidetriphosphates, means used to label (for example, an avidin-enzymeconjugate and enzyme substrate and chromogen if the label is biotin),and the appropriate buffers for reverse transcription, PCR, orhybridization reactions.

Usually, the kits also contain instructions for carrying out themethods.

Method of Preparation

The compounds of Formula I and II can be prepared in a number of wayswell known to those skilled in the art, including both solid phase andsolution techniques. The compounds can be synthesized, for example, bythe methods described below, or variations thereof as appreciated by theskilled artisan. All processes disclosed in association with the presentinvention are contemplated to be practiced on any scale, includingmilligram, gram, multigram, kilogram, multikilogram or commercialindustrial scale.

As discussed in detail above, compounds of Formula I or II can containone or more asymmetrically substituted carbon atoms, and can be isolatedin optically active or racemic forms. Thus, all chiral, diastereomeric,racemic forms and all geometric isomeric forms of a structure areintended, unless the specific stereochemistry or isomeric form isspecifically indicated. It is well known in the art how to prepare andisolate such optically active forms. For example, mixtures ofstereoisomers can be separated by standard techniques including, but notlimited to, resolution of racemic forms, normal, reverse-phase, andchiral chromatography, preferential salt formation, recrystallization,and the like, or by chiral synthesis either from chiral startingmaterials or by deliberate synthesis of target chiral centers.

As will be readily understood, functional groups present can containprotecting groups during the course of synthesis. Protecting groups areknown per se as chemical functional groups that can be selectivelyappended to and removed from functionalities, such as hydroxyl groupsand carboxy groups. These groups are present in a chemical compound torender such functionality inert to chemical reaction conditions to whichthe compound is exposed. Any of a variety of protecting groups can beemployed with the present invention. Preferred protecting groups includethe benzyloxycarbonyl group and the tert-butyloxycarbonyl group. Otherpreferred protecting groups that can be employed in accordance with thepresent invention are described in Greene, T. W. and Wuts, P. G. M.,Protective Groups in Organic Synthesis 3d. Ed., Wiley & Sons, 1991.

The compounds of Formula I, where Y is CR⁵, can be prepared as shown inScheme 1.

In Scheme 1, the pyrrolopyrimidine scaffold4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine, compound 5, can preparedfrom ethyl cyanoacetate and bromoacetaldehyde diethyl acetal in sixsteps (10% yield). R² is introduced by Mitsunobu alkylation of compound5, using either solid-phase or solution-phase chemistry, to formcompound 6. R² substituents can also be introduced by anion alkylationor Michael addition. A4-formyl-3,5-dimethoxyphenoxymethyl-functionalized (PAL) resin is loadedwith an R⁴ appended amine by reductive amination to form compound 7,employing, for example, methylamine, ethylamine, benzylamine or2,4,6-trimethoxybenzylamine or a suitable salt thereof (such as thehydrogen chloride salt). Compounds 6 and 7 are contacted and heated toallow the S_(N)Ar capture of the alkylated scaffold to form compound 8.Using a solid-phase Suzuki coupling employing the appropriate boronicacid and catalyst (such as palladium), R^(1a) is introduced to formcompound 9. Alternatively, a solution-phase Suzuki coupling can beemployed. Compound 9 is then cleaved from the solid support withtrifluoroacetic acid. Scheme 1 can be carried out under similar reactionconditions as a solution-phase synthesis.

When a primary amine at R⁴ is required, a protecting strategy can beemployed. An acid-labile protecting group, such as, for example,2,4,6-trimethoxybenzylamine, is preferred. Acid labile protecting groupsfor R¹ or R² substituents can also be employed. Other suitable acidlabile-protecting groups commonly used in the art can be found in Greeneand Wuts, Protective Groups in Organic Synthesis, 2d ed, John Wiley &Sons, New York, 1991, the disclosure of which is hereby incorporated byreference in its entirety.

The compounds of Formula I and the compounds of Formula II of theinvention can be prepared as shown in Scheme 2. The compounds of FormulaI, where Y is N, can be prepared as generally described in Hanefeld, U.,Rees, C. W., White, A. J. P., Williams, D. J., “One-pot Synthesis ofTetrasubstituted Pyrazoles Proof of Regiochemistry,” J. Chem. Soc.Perkin Trans 1: 1545-1522, 1996, the disclosure of which is incorporatedherein by reference in its entirety. Scheme 2 is also applicable whereZ=halo or Z═NR₃R₄, (wherein R₃ and R₄ are not hydrogen) as demonstratedin Bishop, A. C. Chemical Genetic Approaches To Highly Selective ProteinKinase Inhibitors, Ph.D. Doctoral dissertation, Princeton University,2000, the disclosure of which is incorporated herein by reference in itsentirety.

General Scheme for Solution Phase Synthesis of PyrrolopyrimidineInhibitors

Solution phase synthesis of pyrrolopyrimidine derivatives is carried outusing the general scheme above. Reactions are analogous to thoseemployed during solid-phase synthesis. Mitsunobu alkylation, anionalkylation or Michael addition type reactions can be used to introduceR₂ substituents in the production of 6. S_(N)aryl reaction of 6 withprimary amines, secondary amines or ammonia (R₄═H), yields 9a. Suzukicoupling of an aryl boronic acid or boronic ester yields 10. Asubsequent deprotection step is required when there are protectinggroups as in the case for RB11 synthesis.

General Scheme for Solution Phase Synthesis of RB10 and RB11

tert-Butyl3-(4-chloro-5-iodo-4-aH-pyrrolo[2,3-d]pyrimidin-7(7aH)-yl)propanoate:The Michael addition was performed as follows:4-Chloro-7,7a-dihydro-5-iodo-4-aH-pyrrolo[2,3-d]pyrimidine (5, 3g, 0.11mol) and Cs₂CO₃ (5.25 g, 0.016 mol) were placed within a 250 ml roundbottom flask, and the contents were subject to high vacuum for 20 min.The flask was purged with argon, t-Butylacrylate (30 ml) was added andthe reaction was left to stir at room temperature overnight. Thereaction was quenched with 100 ml 10% aqueous mono-sodium citrate, andthe organic materials were extracted into ethyl acetate (3×100 ml). Thecombined organics were dried with sodium sulfate, and evaporated invacuo to yield a viscous oil. Silica gel chromatography (ethylacetate:hexanes) and evaporation of the requisite fractions yielded 0.7g (17.2% yield) of the desired product as a white solid. ¹H NMR (399.6MHz, CDCl₃) δ 1.38 (9H, s), 2.76 (2H, t, J=6.4 Hz), 4.50 (2H, t, J=6.4Hz), 7.47 (1H, s), 8.59 (1H, s).

tert-butyl3-(5-iodo-4-(methylamino)-4-aH-pyrrolo[2,3-d]pyrimidin-7(7aH)-yl)propanoate:t-Butyl3-(4-chloro-5-iodo-4-aH-pyrrolo[2,3-d]pyrimidin-7(7aH)-yl)propanoatefrom above (0.05 g, 0.12 mmol) was placed in a 15 ml pressure tube. 2Mmethylamine in THF (7 ml) was added and the vessel was sealed and leftto stir overnight. The volatiles were evaporated in vacuo, the resultantmaterial was quenched with 20 ml 10% monosodium citrate, and thesolution was extracted with ethyl acetate (3×20 ml). The combinedorganic extracts were dried with sodium sulfate and evaporated in vacuo.The resultant product (0.069 g, 140% yield) was used without furtherpurification. ¹H NMR (399.6 MHz, CDCl₃) δ 1.38 (s), 2.71 (2H, t, J=6.4Hz), 3.15 (3H, d, J=4.8 Hz), 4.38 (2H, t, J=6.4 Hz), 6.04 (3H, app s),7.04 (s), 8.33 (s).

tert-Butyl3-(5-(3-hydroxyphenyl)-4-(methylamino)-4-aH-pyrrolo[2,3-d]pyrimidin-7(7aH)-yl)propanoate(RB10): t-butyl3-(5-iodo-4-(methylamino)-4-aH-pyrrolo[2,3-d]pyrimidin-7(7aH)-yl)propanoate(123 mmol) from above was placed in a 25 ml round bottom flask,whereupon 3.1 ml dimethoxy ethyleneglycol was added.3-Hydroxyphenylboronic acid (492 mmol pre-dissolved in 0.66 ml ethanol)was added at once, and was followed by 0.5 ml saturated aqueous sodiumcarbonate. Pd⁰(PPh₃)₄ (14 mg, 12 umol) was added to the reaction, thevessel was purged with argon, and set to stir at 80 C overnight. Thereaction was subsequently cooled, and filtered through a bed of celite.The filtrate was evaporated in vacuo, and the residual material wasadhered to silica gel using ethyl acetate as solvent. Silica gelchromatography (ethyl acetate:hexanes) and evaporation in vacuo of therequisite fractions yielded the desired product. MS m/z=369.22.

3-(5-(3-hydroxyphenyl)-4-(methylamino)-4-aH-pyrrolo[2,3-d]pyrimidin-7(7aH)-yl)propanoicacid (RB11): RB10 (9.6 mg, 26 umol) was treated with 2 ml deprotectionsolution (45% TFA, 45% CH₂Cl₂, 5% Me₂S, 5% H₂O) for 1 hr at roomtemperature. The volatiles were evaporated in vacuo, and 1 mlacetonitrile:water:TFA (1:1:0.002) was added. The resultant solution waspurified by reverse-phase HPLC using a linear gradient of water toacetonitrile both containing 0.1% TFA. The requisite fractions werepooled and lyophilized to give the desired product 4.1 mg (79% yield) asa white powder. ¹H NMR (399.6 MHz, d⁶-DMSO) δ 2.85 (2H, t, J=6.4 Hz),3.0 (3H, d, J=4.4 Hz), 4.3, t, J=6.4 Hz), 6.8 (3H, m), 7.27 (1H, app t,J=7.6 Hz), 9.55 (1H, b s) The amino proton resonance was presumablyhidden due to the presence of water in the NMR sample.

General Scheme for Solution Phase Synthesis of RB6

The general solution phase synthetic strategy was used to synthesize RB6in 3 steps from compound 5. RB8 and RB9 were produced in a similarmanner, except benzyamine and dibenzyl amine respectively were usedduring the S_(N)aryl reaction.

Mitsunobu alkylation of 5 with isopropanol: To a dry 50 ml round bottomflask was added 5 (0.5 g, 1.78 mmol) and PPh₃ (0.84 g, 3.2 mmol). Thematerials were dried under high vacuum for 20 m, and the flask waspurged with argon. THF (30 ml) and isopropanol (0.3 ml, 3.9 mmol) wereadded and the flask was cooled in an ethylene glycol/dry ice bathwhereupon DiAD (0.47 g, 2.3 mmol) was added dropwise to the stirredsolution. After 18 h, the volatiles were evaporated in vacuo and theresultant oil was dissolved in ethyl acetate (50 ml) and 50% saturatedsodium bicarbonate (50 ml). The organics were extracted with ethylacetate (3×50 ml), dried with sodium sulfate and evaporated in vacuo toyield an orange oil. Silica gel chromatography (ethyl acetate:hexanes)afforded the desired product as a yellow solid (480 mg, 84% yield). ¹HNMR (399.6 MHz, CDCl₃) δ 1.5 (6H, d, J=6.4 Hz), 5.1 (1H, sp, J=6.8 Hz),7.4 (1H, s), 8.6 (1H, s).

7,7a-Dihydro-5-iodo-7-isopropyl-N-methyl-4-aH-pyrrolo[2,3-d]pyrimidin-4-amine:4-Chloro-7,7a-dihydro-5-iodo-7-isopropyl-4-aH-pyrrolo[2,3-d]pyrimidine(0.3 g, 0.93 mmol) from above was placed within a 15 ml pressure tube. 2M methylamine in THF (15 ml) was added, and the reaction was left tostir overnight. The volatiles were removed in vacuo, and the residue wasdissolved in methanol, 5 ml silica gel were added, and the volatileswere removed in vacuo. The adhered product was purified by silica gelchromatography (ethyl acetate:hexanes), and the requisite fractions werepooled and evaporated in vacuo to yield the desired product (0.25 g, 85%yield). ¹H NMR (399.6 MHz, CDCl₃) δ 1.43 (6H, d, J=6.8 Hz), 3.13 (3H, d,J=4.8 Hz), 5.0 (1H, sp, J=6.8 Hz), 7.02 (1H, s), 8.35 (1H, s).

7,7a-Dihydro-7-isopropyl-N-methyl-5-phenyl-4-aH-pyrrolo[2,3-d]pyrimidin-4-amine(RB6):7,7a-Dihydro-5-iodo-7-isopropyl-N-methyl-4-aH-pyrrolo[2,3-d]pyrimidin-4-amine(0.15 g, 0.475 mmol) from above was placed in a 50 ml round bottomflask, whereupon 12 ml dimethoxy ethyleneglycol was added.3-Hydroxyphenylboronic acid (0.262 g, 1.9 mmol pre-dissolved in 3.3 mlethanol) was added at once, and was followed by 1.9 ml saturated aqueoussodium carbonate. Pd⁰(PPh₃)₄ (55 mg, 47 umol) was added to the reaction,the vessel was purged with argon, and set to stir at 80 C for 48 h. Thereaction was subsequently cooled, and filtered through a bed of celite.The filtrate was evaporated in vacuo, and the residual material wasadhered to silica gel using ethyl acetate as solvent. Silica gelchromatography (ethyl acetate:hexanes) and evaporation in vacuo of therequisite fractions yielded the desired product (94.8 mg, 70.7% yield).¹H NMR (399.6 MHz, d⁶-DMSO) δ 1.76 (6H, d, J=6.8 Hz), 5.03 (3H, d, J=4.8Hz), 5.34 (1H, sp, J=6.4 Hz), 5.53 (1H, q, J=4.8 Hz), 6.73 (1H, m), 6.85(1H, m), 7.25 (1H, app t, J=7.6 Hz), 7.37 (1H, s), 7.59 (1H, s).

Anion alkylation of 5 with methyl iodide: To a dry 50 ml round bottomflask was added 5 (0.2 g, 0.7 mmol) and 15 ml dry acetonitrile. Thereaction was cooled on ice, and NaH (0.026 g, 1.1 mmol) was added atonce. After stirring for 5 m, MeI (0.152 g, 1.07 mmol) was addeddropwise. The reaction was allowed to warm to room temperatureovernight. The volatiles were evaporated, the residue was dissolved inethyl acetate:water, and the organics were extracted with ethyl acetate(3×50 ml). The organics were dried with sodium sulfate and evaporated invacuo. The residue was subject to silica gel chromatography (ethylacetate:hexanes). The requisite fractions were pooled and evaporated toyield a granular solid (0.12 g, 56% yield). ¹H NMR (399.6 MHz, CDCl₃) δ3.87 (1H, s), 7.35 (1H, s), 8.62 (1H, s).

General Scheme for Solution Phase Synthesis of PyrrolopyrimidineInhibitors SD1 Through SD8

Preparation of 3,5-Disubstituted Aryl Borates

3-Substituted anisoles were either purchased from Aldrich (X═CH₃, CF₃,Br) or synthesized from the corresponding phenols (X=isopropyl,t-butyl). Direct borylation was performed according to the generalprocedures described by Miyaura and Hartwig. Ishiyama, T.; et al., J.Am. Chem. Soc., 124: 390-391, 2002; Ishiyama, T.; et al., Angew. Chem.Int. Ed., 41: 3056-3058, 2002. A typical experimental procedure is givenbelow.

3-Trifluoromethyl-5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)anisole.A flame-dried 100 mL Schlenk tube was charged withbis(pinacolato)diboron (350 mg, 1.38 mmol), [Ir(COD)Cl]₂ (12 mg, 0.018mmol), sodium methoxide (5 mg, 0.09 mmol), and4,4′-di-tert-butyl-2,2′-dipyridyl (8 mg, 0.03 mmol). The flask wasevacuated, placed under argon, and 3-trifluoromethylanisole (2.5 mL) wasadded. The flask was restoppered and evacuated (full vacuum, 2 minutes).The flask was sealed under vacuum and maintained at 90° C. (oil bath)for 96 h. Thereafter, the contents were transferred to a round bottomflask with the aid of ethyl acetate and purified by Kugelrohrdistillation. The product, a viscous oil, distills at 120° C.@10 μm.Isolated yield 497 mg (1.65 mmol, 60%). ¹H NMR (400 MHz, CDCl₃) δ 7.62(s, 1H); 7.43 (s, 1H); 7.18 (s, 1H); 3.82 (s, 1H); 1.32 (s, 1H).

General Experimental Procedure for Coupling of 3,5-Disubstituted ArylBorates to Scaffolds

The appropriate aryl pinacol borate (0.1 mmol) and iodinated substrate(0.1 mmol) were dissolved in acetone and transferred to a Schlenk flask.The solvent was evaporated and the flask was charged with Pd(PPh₃)₄ (3mg) and K₃PO₄ (100 mg). The flask was evacuated, placed under argon, andcharged with 5 mL of degassed anhydrous DMF. The resulting solution washeated at 60° C. for 24 h under argon. Water was added and the mixturewas extracted (3×10 mL) with ethyl acetate. The combined organicfractions were washed with water and saturated aqueous NaCl, dried overNa₂SO₄, and evaporated. The remaining material was loaded unto a small(0.5 cm×8 cm) silica gel column and eluted with 1:4 ethyl acetate:hexanesolution. Isolated yields ranged from 50% to 90%.

X═CH₃ ¹H NMR (400 MHz, CDCl₃) δ 8.62 (s, 1H); 7.32 (s, 1H); 6.91 (s,1H); 6.87 (s, 1H); 6.73 (s, 1H); 5.18 (septet, 1H, J=6.8 Hz); 3.82 (s,3H); 2.37 (s, 3H); 1.54 (d, 6H, J=6.8 Hz).

X═CF₃ ¹H NMR (400 MHz, CDCl₃) δ 8.64 (s, 1H); 7.37 (s, 1H); 7.34 (s,1H); 7.23 (s, 1H); 7.11 (s, 1H); 5.19 (septet, 1H, J=6.7 Hz); 3.88 (s,3H); 1.56 (d, 6H J=6.7 Hz).

X═Br ¹H NMR (400 MHz, CDCl₃) δ 8.63 (s, 1H); 7.33 (s, 1H); 7.23 (s, 1H);7.04 (s, 1H); 6.99 (s, 1H); 5.18 (septet, 1H, J=6.8 Hz); 3.83 (s, 3H);1.55 (d, 6H, J=6.8 Hz).

X=tert-butyl ¹H NMR (400 MHz, CDCl₃) δ 8.61 (s, 1H); 7.33 (s, 1H); 7.15(s, 1H); 6.91 (s, 1H); 6.82 (s, 1H); 5.18 (septet, 1H, J=6.8 Hz); 3.81(s, 3H); 1.56 (d, 6H, J=6.8 Hz); 1.35 (s, 9H).

X═CO₂CH₃ ¹H NMR (400 MHz, CDCl₃) δ 8.63 (s, 1H); 7.76 (t, 1H, J=1.4 Hz);7.54 (dd, 1H, J₁=2.4 Hz, J₁=1.4 Hz); 7.36 (s, 1H); 7.26 (dd, 1H, J₁=2.4Hz, J₁=1.4 Hz); 5.18 (septet, 1H, J=6.8 Hz); 3.91 (s, 3H); 3.88 (s, 3H);1.55 (d, 6H, J=6.8 Hz).

General Experimental Procedure for Demethylation of Inhibitors

General Procedure: The anisole derivative (20 mg) was dissolved inmethylene chloride (5 mL) and transferred to an argon flushed Schlenktube. The solution was chilled to 0° C. before 1 mL of a BBr3 solution(1 M in CH₂Cl₂) was added. The mixture was stirred at 0° C. for 2 h.Saturated aqueous NaHCO3 was added, the biphasic mixture was stirred for15 min, extracted with CH₂Cl₂, and the organic extracts were dried overNa2SO4. The solvent was evaporated and the organic residue was purifiedby flash chromatography on silica gel (1:1 hexane:ethyl acetate eluant).Isolated yields were in excess of 80%.

X═CH3; 1H NMR (400 MHz, CDCl₃) δ 8.62 (s, 1H); 7.31 (s, 1H); 6.87 (s,1H); 6.81 (s, 1H); 6.69 (s, 1H); 5.77 (broad singlet, 1H); 5.17 (septet,1H, J=6.7 Hz); 2.34 (s, 3H); 1.54 (d, 6H, J=6.7 Hz).

X═CF3; 1H NMR (400 MHz, CDCl₃) δ 8.66 (s, 1H); 7.40 (s, 1H); 7.28 (s,1H); 7.20 (s, 1H); 7.11 (s, 1H); 5.19 (septet, 1H, J=6.8 Hz); 1.56 (d,6H J=6.8 Hz).

X═Br; 1H NMR (400 MHz, CDCl₃) δ 8.32 (s, 1H); 7.09 (s, 1H); 7.09 (s,1H); 7.00 (s, 1H); 6.65 (s, 1H); 5.27 (broad quart., 1H, J=4.6); 5.03(septet, 1H, J=6.8 Hz); 3.12 (d, 3H, J=4.6 Hz); 1.48 (d, 6H, J=6.8 Hz).

X=tert-butyl; 1H NMR (400 MHz, CDCl₃) δ 8.64 (s, 1H); 7.35 (s, 1H); 7.08(s, 1H); 6.88 (s, 1H); 6.80 (s, 1H); 5.19 (septet, 1H, J=6.7 Hz); 3.19(d, 3H, J=4.6 Hz) 1.52 (d, 6H, J=6.7 Hz); 1.34 (s, 9H).

General Experimental Procedure for Methylamination

General Procedure: Each compound (20 mg) was dissolved in 5 mL of a THFsolution containing methyl amine (1 M) and transferred to an argonflushed 50 mL Schlenk storage tube. The vessel was sealed and heated at60° C. for 24 h. The THF was evaporated and the remainder waspartitioned between ethyl acetate and aqueous bicarbonate solution. Thebiphasic mixture was extracted with ethyl acetate and the combinedorganic extracts were dried over Na₂SO₄. The solvent was evaporated andthe organic residue was purified by flash chromatography on silica gel(1:3 hexane:ethyl acetate eluant). Isolated yields were in excess of80%.

X═CH₃ ¹H NMR (400 MHz, CDCl₃) δ 8.38 (s, 1H); 7.02 (s, 1H); 6.75 (s,1H); 6.75 (s, 1H); 6.71 (s, 1H); 5.67 (broad s, 1H); 5.07 (septet, 1H,J=6.7 Hz); 3.16 (broad d, 3H, J=4.7 Hz); 2.34 (s, 3H) 1.48 (d, 6H, J=6.7Hz).

X═CF₃ ¹H NMR (400 MHz, CDCl₃) δ 8.38 (s, 1H); 7.19 (s, 1H); 7.15 (s,1H); 7.10 (s, 1H); 7.10 (s, 1H); 5.50 (broad s, 1H); 5.09 (septet, 1H,J=6.7 Hz); 3.20 (d, 3H, 4.9 Hz) 1.52 (d, 6H J=6.7 Hz).

X═Br ¹H NMR (400 MHz, CDCl₃) δ 8.64 (s, 1H); 7.35 (s, 1H); 7.19 (t, 1H,J=1.5 Hz); 7.04 (t, 1H, J=2.0 Hz); 6.95 (dd, 1H, J₁=2.0 Hz, J₂=1.5 Hz);5.17 (septet, 1H, J=6.8 Hz); 1.54 (d, 6H, J=6.8 Hz).

X=t-butyl ¹H NMR (400 MHz, CDCl₃) δ 8.42 (s, 1H); 7.06 (s, 1H); 6.96 (s,1H); 6.94 (s, 1H); 6.82 (s, 1H); 5.25 (broad s, 1H) 5.10 (septet, 1H,J=6.7 Hz); 3.81 (s, 3H); 1.55 (d, 6H, J=6.8 Hz); 1.32 (s, 9H).

Other embodiments and uses will be apparent to one skilled in the art inlight of the present disclosures.

EXEMPLARY EMBODIMENTS

Development of potent and selective inhibitors of individual SDR familymembers have the potential to increase the local concentration ofendogenous hormones with important therapeutic benefits such as cortisolas an anti-inflammatory agent (11β-hydroxysteroid dehydrogenase II), orto block production of potent chemoattractants such as prostaglandin E2for blocking colon cancer or metastatic cancer (Carbonyl reductase 1;FIG. 1) (Forrest, G. L. et al., Chem Biol Interact, 129: 21-40, 2000),or for degradation of agents that cause obesity or glucose intoleranceleading to insulin resistant diabetes such as cortisone(11β-hydroxysteroid dehydrogenase I; FIG. 2) (Oppermann, U. C., et al.,Chem Biol Interact, 130-132(1-3): 699-705, 2001). Of particular focushere is the potential for blocking the action of Carbonyl Reductase 1,which is responsible for the reduction of the C-13 keto group of theanthracycline anti-cancer agents (daunorubicin: Cerubidin®, DaunoXome®;doxorubicin: Adriamycin®) (FIG. 3). The reduction of C-13 keto ofadriamycin, inactivates the anti-cancer activity of daunorubicin andproduces a product (daunorubicinol) which is known to be cardiotoxic.Thus, blocking the action of CBR1 in patients treated with adriamycin,would be predicted to enhance the potency of adriamycin's anti-canceractivity and also to reduce the harmful cardiotoxic effects of theadriamycin metabolite (daunorubicinol). Thus, the SDR family membersrepresent an important class of enzymes critical for control of thebiological activity of a wide variety of endogenous and xenobioticscompounds. By designing inhibitors of individual members of this familyof enzymes new therapies for lung cancer, breast cancer, obesity,diabetes, and for improving the activity and decreasing the toxicity ofexisting anti-cancer drugs should be possible.

Microarray studies (67 tumors from 56 patients) show that CBR1 isupregulated in squamous cell lung carcinoma, but not in small cell lungcarcinoma, (FIG. 4; M. E. Garber et al., Proc. Nat. Acad. Sci. USA, 98:13784, 2001) leading to the hypothesis that AB129 kills such cells byinhibiting CBR1.

Inhibitory activity of AB129 has been demonstrated by measuringreduction of menadione to mendadiol by carbonyl reductase 1 (CBR1).AB129 inhibits the CBR1 catalyzed reduction of menadione by NADPH. TheIC₅₀ for AB129 was approximately 5 μM. PP1 did not inhibit CBR1. (FIG.5) At a concentration as high as 16 μM. AB129 is a competitive inhibitorof CBR1, with respect to NADPH (FIG. 6).

Experiments using interfering RNA (RNAi) downregulate CBR1 by inhibitingtranslation of mRNA in A549 lung carcinoma cells and demonstrate thatCBR1 has a role in development of lung cancer. RNAi inhibition of CBR1translation demonstrates a 60 to 70% decrease in viability of A549 lungcarcinoma cells compared to an approximately 50% decrease in viabilityof A549 cells in the presence of AB129. (FIGS. 7 and 8) This suggeststhat inhibition of CBR1 expression in A549 cells decreases cellviability.

Example 1 The SDR Family Member 11β-Hydroxysteroid Dehydrogenase 2(11β-HSD2) Controls the Local Metabolism of Glucocorticoids and DirectlyRegulates Tissue Specific Nuclear Hormone Signaling

Classical small molecule ligand/receptor pairs in biology interact whenboth are present in the same tissue and are structurally complementaryto one another. An important exception to this paradigm is that of themineralcorticoid receptor which binds both cortisol and aldosterone(Funder, J. W., et al., Science, 242: 583-5, 1988). In the kidney, themineralcorticoid receptor regulates K⁺ uptake and water absorption, inresponse to the rennin-angiotensin-aldosterone signaling cascade.However, since blood concentrations of cortisol are 100-1000 foldgreater than aldosterone, the mineralcorticoid receptor must be“protected” from activation by cortisol in order to allow properregulation by aldosterone. This “protective” function is carried out by11β-HSD2 co-localized with the mineralcorticoid receptor, which convertscortisol to cortisone, a steroid hormone which has no binding affinityfor the mineralcorticoid receptor (FIG. 2) (Funder, J. W., et al.,Science, 242: 583-5, 1988). Congenital loss of this enzyme causesapparent mineralcorticoid receptor excess, due to overstimulation of themineralcorticoid receptor by cortisol, bypassing its normal regulationby aldosterone (White, P. C., et al., Endocr Rev, 18: 135-56, 1997).This unusual mechanism of nuclear receptor ligand regulation suggests apotentially new therapeutic approach to treat asthma.

Example 2 Targeting 11β-Hydroxysteroid Dehydrogenase 2 (11β-HSD2) as aPotential Alternative to Synthetic Corticosteroid Treatment of Asthma

Tissue specific metabolism of steroids is an important factor inregulating the properties of endogenous steroids, perhaps drugs whichinhibit these enzymes could effectively regulate the localconcentrations of beneficial endogenous hormones. In asthma, localadministration of an inhibitor of 11β-HSD2 in the lung would blockconversion of the anti-inflammatory steroid, cortisol to inactivecortisone, thus providing a larger concentration of the body's ownanti-inflammatory agent to reduce bronchial swelling in this tissue. Onepotential benefit is that cortisol in the lung would remain regulatableby tissue specific metabolizing enzymes outside of the lung. Syntheticglucocorticoids currently used in asthma therapy are not metabolized bythese enzymes, and thus can show pleiotropic effects in other tissues ifdosages are not controlled (Barnes, P. J. et al., Am Rev Respir Dis,148: S1-26, 1993). Thus, the strategy proposed here, can avoid thecomplications of administration of synthetic corticosteroids to patientswhich can cause high blood pressure, swelling, changes of mood andweight gain, all of which are known functions of cortisol in the body,but which are unwanted side-effects for asthma patients.

The strategy of regulating the metabolism of cortisol through 11β-HSD2inhibition in the lung is less prone to unwanted side-effects thansynthetic glucocorticoid therapy, even though both act throughglucocorticoid receptors which are present throughout the body. Vastlydifferent levels of 11β-HSD2 are expressed in the lung compared to otherorgans, providing an avenue for potent inhibition in the lung withoutsignificant inhibition systemically. 11β-HSD2 is expressed atsignificantly lower levels in the lung compared to the kidney, adrenaland colon (Romero, D. G., et al., J Steroid Biochem Mol Biol, 72: 231-7,2000). One caveat is that only the mRNA levels have been reported whichmay not directly correspond to a difference in 11β-HSD2 protein levels.By administering a relatively low dose of an 11β-HSD2 inhibitor directlyin the lung (intratracheal in the mouse, or with a nebulizer inpatients), a significant inhibition of 11β-HSD2 is achieved in the lung,resulting in a significant increase of cortisol concentration. Any ofthe inhibitor which is absorbed systemically will encounter much largerconcentrations of 11β-HSD2, and thus will be unable to significantlyperturb 11β-HSD2 function in these tissues, resulting in less severeside-effects compared to synthetic glucocorticoid therapy. Thus, thedifferent levels of 11β-HSD2 expression in the lung can provideadditional control over unwanted systemic glucocorticoid stimulation inasthma patients.

Example 3 Evidence Linking 11β-Hydroxysteroid Dehydrogenase 2 (11β-HSD2)Inhibition as a Therapy for Asthma

Schleimer and coworkers have suggested 11β-HSD2 is an attractive targetfor treatment of asthma (Feinstein, M. B. et al., Am J Respir Cell MolBiol, 21: 403-8, 1999). They point out that a natural product isolatedfrom licorice is an ancient therapy for asthma and many otherinflammatory diseases such as eczema and Addison's disease (Persson, C.G., Pulm Pharmacol, 2: 163-6, 1989). The major bioactive component oflicorice, glycyrrhizic acid is in fact an inhibitor of 11β-HSD2 (IC₅₀=8nM) (FIG. 9) (Diederich, S., e al., Eur J Endocrinol, 142: 200-7, 2000).Schleimer and coworkers first confirmed that 11β-HSD2 is present in lungepithelial cells, and that cortisol is rapidly oxidized to cortisone inthis tissue. Next, they confirmed that glycyrrhizic acid'santi-inflammatory activity in cells is dependent on the presence of11β-HSD2, further supporting the link between this natural product and11β-HSD2. Unfortunately, glycyrrhizic acid is not likely to be a verygood asthma therapy because of its non-selective nature. It inhibits11β-HSD1, which blocks production of cortisol from cortisone, resultingin a reduction of anti-inflammatory cortisol concentration, at almostequivalent potency to its inhibition of 11β-HSD1 (IC₅₀=40 nM)(Diederich, S., e al., Eur J Endocrinol, 142: 200-7, 2000). In fact,recent studies with a derivative of glycyrrhizic acid, carbenoxolone,with similar potency and specificity for 11β-HSD1 and 2 (FIG. 2), hasshown potent inhibition of 11β-HSD2 in men with type 2 diabetes(11β-HSD2 inhibition was not measured) (Andrews, R. C., et al., J ClinEndocrinol Metab, 88: 285-91, 2003).

Glycyrrhizic acid's non-selective nature is due to its interaction withthe glucocorticoid binding pocket of 11β-HSD 1 & 2 which is conservedbetween the two enzymes. In fact, both enzymes can operate as areductase or an oxidase, catalyzing both formation and degradation ofcortisol. In the body, the directionality of each enzyme is controlledby regulation of re-dox cofactor concentration with NADPH preferred by11β-HSD1 and NAD⁺ preferred by 11β-HSD2 (Diederich, S., e al., Eur JEndocrinol, 142: 200-7, 2000). A highly selective 11β-HSD2 inhibitor isdeveloped by targeting the NAD⁺ binding pocket of 11β-HSD2 which isdifferentiated from that of 11β-HSD1 by the preference of 11β-HSD2 forNAD⁺ as a cofactor and preference of 11β-HSD1 for NADPH. In fact, asimilar approach has been successfully used to design selectiveinhibitors of 11 β-HSD1, which is an attractive drug target fortreatment of obesity and insulin resistant diabetes (Barf, T., et al., JMed Chem, 45: 3813-5, 2002). The same strategy is applied for treatmentof asthma by targeting 11β-HSD2.

Example 4 Discovery of an Inhibitor of the SDR Family Member, CarbonylReductase 1 (CBR1)

Three structurally similar compounds (AB129, 1, AB60, 3, and AB61, 4),but not PP1, 2 (FIG. 10) were found to cause mild to severe cell killingin the human lung cancer cell line, A549. (FIG. 11) AB129 affects thecell cycle in A549 cells with approximately 12% of A549 cells in G2/Mphase, whereas PP1-treated A549 cells have approximately 5.6% of cellsin G2/M phase. AB129 treated cells show a proportion of cells that maybe polyploid. (FIG. 12). Many small molecules with cell killing activityon cancer cell lines have been described to date (REFs) yet often thetargets of the small molecules cannot be identified because thecompounds bind poorly (>1 μM Kd) to the targets, or the targets areexpressed at very low abundance (<100,000 copies/cell), orderivatization of the small molecule necessary for attachment of anaffinity tag (biotin) or attachment to a bead (for affinity purificationof the target protein) reduces the cellular activity and thus ability tobind the target. A strategy was pursued to identify the target ortargets of the AB129, AB60, and AB61 compounds based on affinitychromatography.

A derivatized form of AB129 was synthesized. The AB129 compound producedthe most potent A549 cell killing response. The derivatized compound wasbound to an agarose bead (P in FIG. 13) and cell lysates were passedover the beads, hoping to retain the true target of AB129 and eliminateall non-interacting proteins. Importantly, a control resin (C in FIG.13) was used to determine if any interacting proteins were trulyspecific for AB129, or were common binders of the pyrazolopyrimidinescaffold. The results of the affinity purification (pull-down)experiment are shown in FIG. 14. This type of approach is successful incases when the target affinity is high (<1 μM) and when the site ofattachment to the bead does not perturb the binding to the cellulartarget (Mayer, T. U., et al., Science, 286: 971-4, 1999; Kwok, B. H., etal., Chem Biol, 8: 759-66, 2001). Typically, hits from forward chemicalgenetic screens are of poor potency (>20 μM), and thus the affinitycapture strategy is unsuccessful.

Using mass spectrometry the proteins retained on the AB129 beads wereanalyzed and three proteins identified, including carbonyl reductase 1(CBR1) (FIG. 15-22). To confirm that CBR1 is inhibited by AB129 CBR1 wasexpressed in bacteria. It was shown that AB129 is a pure NADPHcompetitive inhibitor of CBR1, with a Ki of 400 nM (FIG. 23). This is avery potent compound for an initial hit in a broad based screen.Importantly, the known kinase inhibitor, PP1, (FIG. 5) does not inhibitCBR1, in this in vitro assay, nor does CBR1 bind to control PP1-agarosebeads, used as a control for affinity purification of the targets ofAB129 (FIG. 14).

To understand the basis for the potency of AB129 against CBR1, acomputer algorithm was used for molecular docking of AB129 to anavailable crystal structure of porcine CBR1 to produce a model of thebound structure of AB129 (FIG. 24). This modeled co-structure wasexperimentally validated by site-directed mutagenesis of multiple aminoacids in the proposed AB129 binding pocket (including a conserved Asnresidue common to all SDR family members—FIG. 25-27) and identificationof AB129 resistant mutants of CBR1 (FIG. 28-30). This binding model alsopotentially explains the importance of the hydroxyl moiety attached tothe phenyl ring of AB129, as being critical for a H-bonding interactionwith an active site Asn. Since PP1 lacks this key hydroxyl group, thismodel potentially explains the structure activity relationshipdifference between PP1 and AB129 in terms of CBR1 inhibition. Aconclusion from these data is that AB129 is a potent inhibitor of CBR1,a member of the SDR family of enzymes, which are responsible for anumber of important small molecule metabolic steps in a variety oforgans and cell types.

GSH modified prostaglandins were recently discovered in colorectalcancer cells. (FIG. 31; Biochim Biophys Acta 1584: 37-45, 2002) CBR1 hasa glutathione binding site distinct from the AB129 binding site.Glutathione binding activity of wild type and N90V mutant CBR1 wastested. The results demonstrate that glutathione binding activity isseparate from the AB129 binding activity as demonstrated for the N90Vmutant CBR1. FIG. 32. These results indicate that a mechanism exists forinhibition of CBR1 and prostaglandin synthesis which can be effective ininhibition of proliferation of colorectal cancer cells.

Example 5 Does Inhibition of CBR1 with AB129, Increase the Cell KillingPotency of Daunorubicin?

One important cellular function of CBR1 is to metabolize xenobioticssuch as the anticancer agent daunorubicin, a member of the anthracyclinantibiotic agents including adriamycin. Experiments were performed totest whether daunorubicin and AB129 treated cells were capable ofexhibiting cell toxicity at concentrations lower than that needed foreach individual compound to induce cell death alone. In agreement withthis therapeutic strategy, A549 cells were treated with 0.4 μM of AB129which led to a 15% loss of viability after two days of treatment (FIG.33-34). Similarly, daunorubicin, as a single agent, when added to A549cells at 0.8 μM let to a similar 15% loss of viability of A549 cells.However, when the two drugs, AB129 and daunorubicin were added incombination at 0.4 μM and 0.8 μM, respectively, a decrease in almost 70%of A549 cell viability was observed (FIG. 33-34). This experimentsuggests that AB129 is capable of enhancing the potency of daunorubicinmediated cancer cell killing. Moreover, since AB129 does this throughinhibition of CBR1, the toxic metabolite of daunorubicin, daunorubicinolis not produced and thus in vivo the cardiotoxic effects ofdaunorubicinol, or other anthracyclin anti-cancer therapy should beenhanced.

Example 6 Other Targets of AB129, Including Potential Protein Kinases

AB129 is an analog of PP1, the Src family protein kinase inhibitor.Experiments were performed to determine whether AB129 was capable ofinhibiting the Src family kinase, Fyn. In fact AB129 is a potent (10 nMIC₅₀) inhibitor of Fyn. While the ability of AB129 to inhibit proteinkinases as well as CBR1 could be important for its biological activityin some settings, AB129 compound was modified to produce a pure CBR1inhibitor. Such a compound would serve as a test compound fordetermining the importance of dual inhibition of CBR1 and proteinkinases. The crystal structure of PP1 bound to Hck, a Src family kinase,shows that the exocyclic amine of PP1 makes a key H-bond interactionwith a back-bone carbonyl group of Hck in the ATP binding pocket. It waspredicted that addition of a methyl group to this amine of AB129 woulddisrupt this H-bond interaction because it would point away from thephenyl ring, thus eliminating the H-bond donor of AB129. In fact, theresulting analog of AB129, RB6 (FIG. 35) was found to be equipotent as aCBR1 inhibitor, yet is predicted to be >100 fold less potent as aninhibitor of Fyn. Anti-Fyn IC₅₀ for RB6 was 70 μM. Anti-Fyn IC₅₀ forAB129 was 10 nM The ability to generate inhibitors for protein kinases(PP1), or CBR1 (RB6), or both targets (AB129) (FIG. 35), should help indistinguishing between the cellular effects of CBR1 and/or kinaseinhibition.

Example 7 Designing New Potent and Selective Inhibitors of SDR FamilyMembers Including Carbonyl Reductase 1 (CBR1), and 11β-HydroxysteroidDehydrogenase 1 and 2 (11β-HSD1 and 2)

Incorporating SAR data obtained from a small set of synthesizedcompounds, and allowing as yet untried diversity elements, a library ofputative CBR inhibitors was envisioned that conserves the putativepharmacophore but introduces structural diversity elements that mightincrease the affinity and specificity of the library members towardvarious SDR enzymes. The proposed library utilizes a pyrrolopyrimidinescaffold as opposed to the pyrrazolopyrimidine scaffold of AB129, andthe compounds synthesized thus far indicate the anti-CBR activity ofboth of these scaffold types are comparable. The docked AB129-CBRstructure was used to inform the choice of library substituentsindicated in FIG. 36.

Diversity element R³ or R⁴ (Formula I) include either proton or alkylsubstituents. Compounds with H-bond donors at this position can inhibitkinases, as does AB129, so the presence of alkyl substituents at thisposition should shift affinity away from kinases. AB129 and the analogssynthesized thus far possess saturated alkyl substituents at R². Inaddition to such substituents, the library will also include negativelycharged substituents or planar aromatic groups at this position. Thedocking model indicates the t-butyl group (analogous to the position ofR²) of AB129 is solvent exposed, and in close proximity to one lysineand two arginine residues forming the binding cavity for the NADP(H)phosphate. To elicit potential ion-pairing interactions, negativelycharged groups like p-cyclohexanoic acid will be included. Also adjacentto the t-butyl group of AB129 is the NADP(H) adenine binding cavity. Asubstituent at R² favors an orientation allowing access to this cavity.Thus, planar aromatic substituents such as indole could be accommodatedresulting in increased affinity. Diversity elements at R¹ will includesubstituted phenyl, indole and thiophene moieties selected for potentialH-bonding and charge interactions with specific active site residues.Substituents larger than the AB129 phenoxy gain additional affinity byexploiting van der Walls interactions deeper within the NADPH bindingchannel. Although all H-bond interactions predicted between AB129 andCBR are made to conserved residues, the binding orientation and adjacentresidue identity can vary throughout the SDR family. These interactionscan be of particular interest for tailoring the specificity of thesecompounds to different enzymes of the SDR class.

As mentioned above, it was postulated that both pyrazolopyrimidines likeAB129 and analogous pyrrolopyrimidines would have comparable anti-CBRactivity. In order to verify this assumption, the pyrazolopyrimidine RB2and the analogous pyrrolopyrimidine RB5 were synthesized (FIG. 37).These compounds employ an R² isopropyl as opposed to the AB129 t-butylbecause the Mitsunobu reaction used for RB5 synthesis is not amenable tot-butyl alkylation. Both of these compounds inhibit CBR with similaraffinity (IC₅₀ values comparable to AB129) and kill adenocarcinoma cellsin culture.

The library of pyrrolopyrimidines well suited for SDR inhibition isconstructed using the following solution and solid-phase reactions (FIG.38). The pyrrolopyrimidine scaffold4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine, 5, was chosen for itssynthetic utility and literature precedent. This scaffold has beensynthesized previously (Pudlo, J. S., et al., J Med Chem, 33: 1984-92,1990. Haslam, R., in U.K. Patent 812,336. 1956: U.K), and wassynthesized in our laboratory from ethyl cyanoacetate andbromoacetaldehyde diethyl acetal in six steps (10% yield). The librarysynthesis will involve introduction of R² by Mitsunobu alkylation of thescaffold, 5, using solution-phase chemistry, and a resin will be loadedwith an R³ or R⁴ appended primary amine by reductive amination.Combining these materials and heating will allow S_(N)Ar capture of thealkylated scaffold. Finally, a solid-phase Suzuki coupling to introduceR³ and TFA mediated cleavage should yield the library members. Similarreaction conditions and the use of the scaffold, 5, are also amenable tosolution-phase syntheses.

Primary amines containing diversity element R¹ will be coupled to4-formyl-3,5-dimethoxyphenoxymethyl-functionalized (PAL) resin to yield7 in a manner analogous to published conditions (Moon, H. S., et al.,Journal of the American Chemical Society, 124: 11608-11609, 2002). Whena primary amine at R³ or R⁴ is required, a protecting strategy can beemployed. Thus, acid-labile 2,4,6-trimethoxybenzylamine should besuitable for this use. This amine mirrors the structure of thefunctionalized resin and should be equally acid sensitive during thecleavage reaction. Separately, in solution phase, diversity element R²will be introduced by Mitsunobu alkylation (Ding, S., et al., J OrgChem, 66: 8273-6, 2001) of 5 to produce 6. Although Mitsunobu alkylationhas been demonstrated on solid phase (Ding, S., et al., J Am Chem Soc,124: 1594-6, 2002. Ding, S., et al., J Comb Chem, 4: 183-6, 2002), 6 wasprepared in solution. A similar coupling and reductive aminationstrategy using a purine scaffold has been developed for the synthesis ofderivatized purines used as kinase inhibitors (Ugarkar, B. G., et al., JMed Chem, 43: 2894-905, 2000). In parallel, each of the resultantproducts 6 will be reacted with the resin bound amine 7 to yieldcompound 8 on solid support. Again in a manner analogous to that usedfor the preparation of kinase inhibitors, 8 will be treated withcommercially available boronic acids using Suzuki coupling conditions tointroduce the diversity element R¹. Similar reactions have been carriedout in solution phase (Ugarkar, B. G., et al., J Med Chem, 43: 2894-905,2000). The compounds can then be cleaved from the PAL resin usingtrifluoroacetic acid.

Optimizations of both reductive amination and scaffold loading (FIG. 20)have been performed using a number of conditions, and good results havebeen obtained. The reductive amination reactions to produce 7 haveutilized methylamine, ethylamine, benzylamine and2,4,6-trimethoxybenzylamine (FIG. 39). The reactions were carried out inpeptide synthesis cartridges using the reducing agent NaBH(OAc)₃ in amanner analogous to that previously reported (Moon, H. S., et al.,Journal of the American Chemical Society, 124: 11608-11609, 2002). Whenusing 2,4,6-trimethoxybenzylamine the HCl salt was used, and astoichiometric quantity of DIEA was also included. Comparable yieldswere obtained using 5 and 20 eq. (0.1 and 0.4 M respectively) of aminewith either THF or 1:1 THF:DMF as evidenced by FMOC quantitation (Bunin,B., The Combinatorial Index, ed. San Diego: Academic Press, 1998).

Loading of the scaffold with resin bound primary amine 7 was alsoattempted using each of the amine-loaded resins. Either THF or n-BuOH at60 or 90° C. respectively in the presence of 10% DIEA was employed. Thevalues reported (FIG. 39) represent conversion at 90° C. for 18 h, asdetermined by FMOC quantification.

The conditions for both reductive amination and resin loading appear towork well for the conditions tested.

The pyrrolopyrimidine scaffold 5 was also used as starting material forthe solution phase synthesis of RB5 and RB6 (FIG. 10) with greater than50% overall yield. Thus, should library members demonstrate activity invitro and larger quantities of material are needed, a solution-phasestrategy can be more efficient. RB5 synthesis commenced with Mitsunobualkylation of 5 with 2-propanol resulted in the preparation of $ ingreater than 90% yield. Mitsunobu reactions of pyrrolopyrimidines arescarce in the literature; however, the utility of the transformation hasbeen demonstrated with purines. Therefore, an analogous procedure usingDiAD, PPh₃ and 2-propanol was employed (Ding, S., et al., J Org Chem,66: 8273-6, 2001). Compound 6 was subsequently aminolyzed at elevatedtemperature in a sealed vessel using a saturated methanolic ammoniasolution. As expected, this reaction was selective for the 4-chloroposition of 6 for both ammonia and methylamine used during the synthesisof RB5 and RB6 respectively. Treatment of the products with3-(hydroxyphenyl)boronic acid afforded RB5 and RB6 using availableSuzuki coupling conditions (Moon, H. S., et al., Journal of the AmericanChemical Society, 124: 11608-11609, 2002). These conditions do notrequire the protection of hydroxylic or amino substituents of theboronic acids.

In vitro assays for the measurement of CBR activity have been developed(Bohren, K. M., et al., J Mol Biol, 244: 659-64, 1994) and used in ourlaboratories. CBR was expressed in E. coli and purified usingglutathione beads; CBR has a naturally occurring glutathione bindingsite. An N-terminal 6-His tagged protein was prepared to allowpurification by metal affinity. Our CBR assay employs the syntheticsubstrate Menadione (2-methyl-1,4-naphthoquinone). Reaction progress ismonitored by the decrease in NADPH absorbance at 340 nm. Saturatingconcentrations of Menadione with variable concentrations of NADPH areemployed in order to ascertain K_(I) values.

A high-throughput assay will be optimized for analyzing librarycompounds. A 96-well format will be employed, and the disappearance ofNADPH will be monitored either by fluorescence or absorbance. Usingfixed substrate and enzyme concentrations, IC₅₀ values for librarymembers can be obtained. Cell culture assays for CBR inhibition can alsobe developed, as CBR inhibitors in the presence of daunorubicin would beexpected to lead to a further decrease in cell proliferation rate thaneither compound alone.

In addition to the use of CBR activity for screening library compounds,recombinant 11β-HSD1 is prepared (Nobel, C. S., et al., Protein ExprPurif, 26: 349-56, 2002). This enzyme is a membrane-bound glycoprotein,and previous reports indicate that the isolation of active enzyme is nottrivial. An expression system using Pichia pastoris has been described.A yeast expression vector for 11β-HSD1 incorporating an N-terminal 6-Histag and a picornavirus protease cleavage site. Followingcharacterization of the enzyme activity, an assay similar to that usedfor detecting CBR activity is developed. An expression system for17β-HSD1 is developed to allow the specificity of these compounds to befurther investigated.

The inhibitor screening results should provide valuable SAR data for thepyrrolopyrimidine pharmacophore, and provide valuable information forproducing even more effective inhibitors in the future. Continuingstudies to assess the selectivity of these analogs among different SDRenzymes and associated cellular phenotypes will be pursued. Differentialactivity of the library members toward CBR and other SDR members whencoupled with available crystallographic and sequence data, should helpto identify important structural and electronic features that lead toeffective and specific inhibition of SDR enzymes.

Chemical genetic screens for small molecules which target diseaserelated biological processes hold great promise for development offuture medicines. A well designed chemical genetic screen like a welldesigned genetic screen requires manipulation of the pathway of interestsuch that early-low potency “hits” can be identified. This precludes thesimultaneous screening of more than one pathway in each assay. Since alimited portion of chemical space is probed in any library of smallmolecules, there is a limited chance that a potent and selective agenttargeting a pathway of interest will be present. Consequently thefrequency of identifying true drug-leads in such screens has beenrelatively low. Chemical-genetic screens have an additional challenge,compared with genetic screens, that of target identification.Identification of the target of a small molecule lead compound isdifficult because the affinity of such early hits are often low, andthus not amenable to successful affinity purification strategies whichrequire tight, or irreversible inhibitors (usually only found in naturalproducts or advanced drug development candidates). To overcome theseproblems, the traditional format of chemical genetic screens wasinverted. Rather than screening a very large collection of smallmolecules for antagonists or agonists of a single pathway, a small panelof compounds was screened for the ability to perturb any pathway in apanel of cell lines with high potency and selectivity. A strategy wasexploited utilizing a cell morphology-microscopy based assay coupledwith an automated image analysis algorithm designed to detectperturbations to a great many cell processes simultaneously including,for example, cell cycle arrest point, cytoskeletal structure, celladhesion status, organelle organization. This approach allowedphenotypic effects of all members of the library to be scored, andshowed that almost every compound in the library at the highest dosesanalyzed (10 μM), produced some phenotypic effects. AB129 was selected,which potently produced a novel phenotype, (several controls wereincluded such as, Taxol and K252a, to define known phenotypesignatures), in a single cell line—the lung cancer A549 line, but notother cell lines. Using traditional target identification methodsapplied to natural products, but rarely applied to first generation hitsfrom chemical genetic screens the target of AB129 in A549 lysates wasidentified as an NADPH dependent reductase, carbonyl reductase 1 (CBR1).CBR1 serves a dual role of prostaglandin biosynthesis and xenobioticmetabolism. In vitro assays demonstrated AB129 is an NADPH competitiveinhibitor of CBR1 with a Ki of approximately 300 to 400 nM, validatingthe overall approach to be successful at identification of potent leadcompounds. The relevance of CBR1 to lung cancer was explored throughanalysis of transcriptional profiling data of various lung cancer celllines. This analysis revealed CBR1 to be a highly upregulated transcriptin adenocarcinomas suggesting it might play an important role inproducing prostaglandins as autocrine factors for A549 cell survival.siRNA studies confirm that CBR1 is essential for A549 cell viability,confirming the mode of action of AB129 at inducing A549 cell death. Toindependently confirm the ability of AB129 to inhibit CBR1 in A549cells, an assay based on the role of CBR1 in attenuating the anti-canceraction of daunorubicin was employed. Indeed, AB129 is able to potentiatedaunorubicin action in A549 cells, suggesting the former can be anattractive combination therapy with daunorubicin. Moreover, AB129 isable to block production of the cardiotoxic metabolite daunorubicinolfrom daunorubicin. Thus, a new broad based phenotype profiling methodallowed for system wide screening of chemical libraries allowed for thediscovery of a potent small molecule capable of selective killing oflung cancer A549 cells and potentiating the action of daunorubicin.

Example 8 New Potent and Selective Inhibitors of SDR Family MembersIncluding Carbonyl Reductase 1 (CBR1), and 11β-HydroxysteroidDehydrogenase 1 and 2 (11β-HSD1 and 2)

Compound RB8 employs a substituent, benzyl, at the exocyclic amine onthe pyrrolopyrimidine/pyrazolopyrimidine scaffold. The anti-CBR IC₅₀=4.4μM, and the anti-Fyn IC₅₀=20 μM.

Compound RB11 employs a carboxy alkyl substituent at N-9 of thepyrrolopyrimidine/pyrazolopyrimidine scaffold. Compound RB11demonstrates an improved anti-CBR IC₅₀ activity. An increased affinitycan be attributed to potential hydrogen bond interactions between thecarboxylate and charged residues including Asn 13, Arg 41, and Arg 37 ofCBR. These residues would otherwise interact with the NADPH 3′-OPO₃ ²⁻phosphate upon substrate binding, and can provide specificity for shortchain dehydrogenase/reductase (SDR) utilizing NADP(H) rather thanNAD(H). The anti-CBR IC₅₀ for compound RB11 is 220 nM.

RB10 is an intermediate in the synthesis of RB11. The anti-CBR IC₅₀=1.15μM. An improved IC₅₀ for compound RB10 may be due to its inability tohydrogen bond to residues including Asn 13, Arg 41, and Arg 37 of CBR1.

Substituents at N-9 of the pyrrolopyrimidine/pyrazolopyrimidine scaffoldcan further include alkyl chains substituted with carboxyl and/orphosphoryl, e.g., R₂ substituents of compounds of Formulas I, II, orIII.

In addition to the above compounds, the effect of substituents at themeta position of the phenyl ring have been studied (see below, CompoundA, as an example of a compound derived from Formula I). Potency asanti-CBR activity can be increased with electron withdrawing groups (Br,CF₃) at the 5-position of the pyrrolopyrimidine/pyrazolopyrimidinescaffold. The CBR binding can be tolerant of even large substituents(tert-butyl) at this position.

Substituting a halo substituent, for example, chloro or bromo, in placeof the exocyclic amine (see below, Compound B, as an example of acompound derived from Formula III) provides an increased affinity forCBR binding. Compounds of the present invention can employ an exocyclicamine or a halo substituent as part of thepyrrolopyrimidine/pyrazolopyrimidine scaffold. Substituting a chlorosubstituent for a methylamino substituent on thepyrrolopyrimidine/pyrazolopyrimidine ring gives a roughly 10-foldincrease in potency. For substituents of chloro or bromo, the IC₅₀ isapproximately 30 nM.

Example 8 New Potent and Selective Inhibitors of SDR Family MembersIncluding Carbonyl Reductase 1 (CBR1), and Src Family Protein Kinase,Fyn

A chloro substituent in place of the exocyclic amine on thepyrrolopyrimidine/pyrazolopyrimidine scaffold provides increasedaffinity. See SD1 and SD5 below. The switch from methylamino to chlorosubstituents on the pyrimidine ring gives a roughly 10-fold increase inpotency in all cases. For SD1 compound, having methylamino and bromosubstituents, the anti-CBR IC₅₀ is 220 nM. For SD5 compound, havingchloro and bromo substituents, the anti-CBR IC₅₀ is 27 nM.

Because replacement of the exocyclic amine with a more hydrophobic,electron-withdrawing substituent (Cl) increases potency, these resultssuggest that exocyclic methylamino can be substituted with halogens (F,Cl, Br, I), hydrogen, small electron-withdrawing groups (NO₂, CN, etc.),or small alkyl and haloalkyl groups at this position.

In addition the effect of substituents at the meta position of thephenyl ring increases potency as a CBR1 inhibitor (see below). Potencyas a CBR1 inhibitor increases with electron withdrawing groups (Br, CF₃)at the 5-position and the CBR seems tolerant of even large substituents(t-butyl) at this position. substituents at the meta position includeelectron withdrawing groups, for example, ester and amide linkages(—COOR, —CONHR).

The anti-CBR IC₅₀ for SD2 is 3.04 μM. The anti-CBR IC₅₀ for SD6 is 193nM

The anti-CBR IC₅₀ for SD3 is 7.65 μM. The anti-CBR IC₅₀ for SD7 is 376nM.

The anti-CBR IC₅₀ for SD4 is 416 nM. The anti-CBR IC₅₀ for SD8 is 67 nM.

Example 9 New Potent and Selective Inhibitors of SDR Family MembersIncluding Carbonyl Reductase 1 (CBR1), and Src Family Protein Kinase,Fyn

Table 1 shows compounds of the present invention that are inhibitors ofthe Src family kinase, Fyn, and are inhibitors of carbonyl reductase 1(CBR1). AB129 is a potent (10 nM IC₅₀) inhibitor of Fyn. While theability of AB129 to inhibit protein kinases as well as CBR1 could beimportant for its biological activity in some settings. AB129 compoundwas further modified to produce compounds of the present invention whichare CBR1 inhibitors with reduced inhibitory activity for Fyn.

TABLE 1 IC₅₀ for anti cFYN and anti-hCBR1 IC₅₀ anti- Compound StructureIC₅₀ anti-c-Fyn-wt hCBR1

110 nM >20 μM

50 nM >20 μM

50 nM >20 μM

8 nM 790 nM

50 nM >20 μM

5 μM >20 μM

0.2 μM 930 nM

3 nM 1 μM

1.2 μM >20 μM

11 nM 1 μM

~20 μM >20 μM

120 nM >20 μM

12 nM 760 nM

70 μM 590 nM

not determined (n/d) >20 μM

20 μM 4.4 μM

n/d >20 μM

n/d 1.15 μM

n/d 220 nM

n/d 220 nM

n/d 3.04 μM

n/d 7.65 μM

n/d 416 nM

n/d 28 nM

n/d 193 nM

n/d 376 nM

n/d 67 nM

When ranges are used herein for physical properties, such as molecularweight, or chemical properties, such as chemical formulae, allcombinations and subcombinations of ranges and specific embodimentstherein are intended to be included.

The disclosures of each patent, patent application and publication citedor described in this document are hereby incorporated herein byreference, in their entirety.

Those skilled in the art will appreciate that numerous changes andmodifications can be made to the embodiments of the invention and thatsuch changes and modifications can be made without departing from thespirit of the invention. It is, therefore, intended that the appendedclaims cover all such equivalent variations as fall within the truespirit and scope of the invention.

What is claimed is:
 1. A compound of Formula I:

or a pharmaceutically-acceptable salt or prodrug thereof; wherein: Y isN or CR₅; Z is NR₃R₄, halo, H, OH, alkyl, alkyloxy, or haloalkyl; R_(1a)is heteroaryl, or R_(1a) is aryl substituted with at least one of OH,C(═O)NR₆R₇, CN, NO₂, C(═O)OH, C(═O)O-alkyl, (C₁-C₄)alkyl, halo,haloalkyl, or haloaryl; R₂ is C₁-C₆ alkyl or C₄-C₇ cycloalkyl, whereinsaid alkyl or said cycloalkyl is optionally substituted with mono- ordi-alkoxy, mono- or di-halophenyl, mono- or di-(C₁₋₄)alkoxy phenyl,mono- or di-(C₁₋₄)alkyl phenyl, perhalo(C₁₋₄)alkyl phenyl, carboxyl,tert-butyl carboxyl, phosphoryl, (C₁₋₆)alkyl, (C₄₋₇)cycloalkyl, indolyl,isoindolyl, pyridyl, naphthyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl,pyrimidinyl, pyrazinyl, pyridazinyl, furyl, thienyl, or alkylmorpholino;R₃ and R₄ are independently H, C₁-C₆ alkyl, t-Boc,morpholino(C₁-C₄)alkyl, carboxy(C₁-C₃)alkyl,(C₁-C₄)alkoxycarbonyl(C₁-C₃)alkyl, aryl, heteroaryl, aryloxy,heterocycle, cycloalkyl, alkenyl with the proviso that the double bondof the alkenyl is not present at the carbon atom that is directly linkedto N, alkynyl with the proviso that the triple bond of the alkynyl isnot present at the carbon atom that is directly linked to N,perfluoroalkyl, —S(O)₂alkyl, —S(O)₂aryl, —(C═O)heteroaryl, —(C═O)aryl,—(C═O)(C₁-C₆) alkyl, —(C═O)cycloalkyl, —(C═O)heterocycle,alkyl-heterocycle, aralkyl, arylalkenyl, —CONR₆R₇, —SO₂R₆R₇,arylalkoxyalkyl, arylalkylalkoxy, heteroarylalkylalkoxy, aryloxyalkyl,heteroaryloxyalkyl, aryloxyaryl, aryloxyheteroaryl, alkylaryloxyaryl,alkylaryloxyheteroaryl, alkylaryloxyalkyamine, alkoxycarbonyl,aryloxycarbonyl, or heteroaryloxycarbonyl; R₅ is independently H, —OH,halo, optionally monosubstituted (C₁-C₆)alkyl, optionallymonosubstituted (C₁-C₄)alkoxycarbonyl, optionally monosubstituted(C₁-C₄)alkanoyl, carbamoyl, optionally monosubstituted (C₁-C₄)alkylcarbamoyl, phenyl, halophenyl, optionally monosubstituted(C₁-C₄)alkylphenyl, optionally monosubstituted (C₁-C₄)alkoxyphenyl, oroptionally monosubstituted perhalo(C₁-C₄)alkylphenyl, wherein saidoptional substitution is (C₁-C₄)alkyl, OH, or halogen; R₆ and R₇ areindependently H, alkyl, aryl, heteroaryl, alkylaryl, arylalkyl,heteroarylalkyl, or alkylheteroaryl; provided the compound is not1-tert-butyl-3-p-tolyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine.
 2. Acompound according to claim 1, wherein Y is N.
 3. A compound accordingto claim 2, wherein R_(1a) is heteroaryl.
 4. A compound according toclaim 3, wherein R_(1a) is pyrryl, furyl, pyridyl, 1,2,4-thiadiazolyl,pyrimidyl, thienyl, isothiazolyl, imidazolyl, tetrazolyl, pyrazinyl,pyrimidyl, quinolyl, isoquinolyl, thiophenyl, benzothienyl,isobenzofuryl, pyrazolyl, indolyl, purinyl, carbazolyl, benzimidazolyl,or isoxazolyl.
 5. A compound according to claim 4, wherein R_(1a) isbenzimidazolyl.
 6. A compound according to claim 1, wherein R₂ is2-methyl-propane.
 7. A compound according to claim 1, wherein R₃ and R₄are H.
 8. A pharmaceutical composition, comprising: a pharmaceuticallyacceptable carrier; and a compound according to claim
 1. 9. Apharmaceutical composition according to claim 8, further comprising atleast one anthracycline compound.
 10. A pharmaceutical compositionaccording to claim 9, wherein said anthracycline compound isdaunorubicin, doxorubicin, epirubicin, idarubicin, or a mixture thereof.11. A method for treating a disease in a mammal in need thereof,comprising the steps of: administering to the mammal a compositioncomprising an effective amount of a compound according to claim
 1. 12.The method of claim 11, wherein Y is N.
 13. The method of claim 12,wherein R_(1a) is heteroaryl.
 14. The method of claim 13, whereinpyrryl, furyl, pyridyl, 1,2,4-thiadiazolyl, pyrimidyl, thienyl,isothiazolyl, imidazolyl, tetrazolyl, pyrazinyl, pyrimidyl, quinolyl,isoquinolyl, thiophenyl, benzothienyl, isobenzofuryl, pyrazolyl,indolyl, purinyl, carbazolyl, benzimidazolyl, or isoxazolyl.
 15. Themethod of claim 14, wherein R_(1a) is benzimidazolyl.
 16. The method ofclaim 11, wherein the disease is cancer, inflammation, or metabolicdisease.
 17. The method of claim 16, wherein the disease is coloncancer, ovarian cancer, leukemia, lymphoma, myeloma, acute myeloidleukemia, acute lymphocytic leukemia, chronic myelogenous leukemia,neuroblastoma, breast cancer, acquired immunodeficiency syndrome(AIDS)-associated Kaposi's sarcoma (KS), or lung cancer.
 18. The methodof claim 16, wherein the disease is diffuse inflammation, focalinflammation, croupous inflammation, interstitial inflammation,obliterative inflammation, parenchymatous inflammation, reactiveinflammation, specific inflammation, toxic inflammation or traumaticinflammation.
 19. The method of claim 16, wherein the disease is obesityor diabetes.
 20. A method for identifying a therapeutic cancertreatment, comprising the steps of: (i) contacting a tumor cell culturewith an effective amount of a compound according to claim 1; (ii)measuring growth inhibition of the tumor cells in culture; and (iii)identifying a therapeutic cancer treatment for a mammalian subject byinhibition of the tumor cell growth in culture.